Insect inhibitory toxin family active against hemipteran and/or lepidopteran insects

ABSTRACT

The present invention discloses a genus of insect inhibitory proteins that exhibit properties directed to controlling Lepidopteran and/or Hemipteran crop pests, methods of using such proteins, nucleotide sequences encoding such proteins, methods of detecting and isolating such proteins, and their use in agricultural systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/441,436, filed Apr. 6, 2012, now U.S. Pat. No. 9,238,678 issuedJan. 19, 2016, which claims priority to U.S. Provisional ApplicationSer. No. 61/472,865 filed Apr. 7, 2011, all of which are incorporatedherein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The file named “P34309US02 Seq.txt” contains the Sequence Listing thatwas created on Nov. 18, 2015. This file is 140,564 bytes (measured in MSWindows), is contemporaneously filed by electronic submission (using theUnited States Patent Office EFS-Web filing system), and is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of insectinhibitory proteins. In particular, the present invention relates toproteins exhibiting insect inhibitory activity against agriculturallyrelevant pests of crop plants and seeds, particularly Lepidopteranand/or Hemipteran species of insect pests.

BACKGROUND OF THE INVENTION

Insect inhibitory proteins derived from Bacillus thuringiensis (Bt) areknown in the art. These proteins are used to control agriculturallyrelevant pests of crop plants by spraying formulations containing theseproteins onto plants/seeds or by expressing these proteins in plants andin seeds.

Only a few Bt proteins have been developed for use in formulations or astransgenic traits for commercial use by farmers to control Coleopteranand Lepidopteran pest species, and no Bt proteins have been used forcommercial control of Hemipteran pest species. Certain Hemipteranspecies, particularly Lygus bugs, are pests of cotton and alfalfa, andtypically are only controlled using broad spectrum chemistries, e.g.,endosulfan, acephate, and oxamyl, which can persist and harm theenvironment. However, dependence on a limited number of these Btproteins can result in occurrence of new pests resistant to theseproteins, and reliance on broad-spectrum chemistries can harm theenvironment.

Hence, there is a continuous need for the discovery and commercialdevelopment of new proteins active against pests of crop plants.

SUMMARY OF THE INVENTION

The present invention provides a novel group, i.e. a new genus, ofinsect inhibitory polypeptides (toxin proteins) which are shown toexhibit inhibitory activity against one or more pests of crop plants.Each of the proteins can be used alone or in combination with each otherand with other Bt proteins and toxic agents in formulations and inplanta, thus providing alternatives to Bt proteins and insecticidechemistries currently in use in agricultural systems.

Recombinant polypeptides are provided which exhibit insect inhibitoryactivity against Hemipteran and/or Lepidopteran pest species, whichoptionally:

-   -   (a) exhibits at least from about 47% to about 100% amino acid        sequence identity, or any percentage point between 47% and 100%,        to one or more of the proteins having the amino acid sequence as        set forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ        ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,        SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24;    -   (b) exhibits at least from about 56% to about 100% amino acid        sequence identity, or any percentage point between 56% and 100%,        to one or more of the proteins having the amino acid sequence as        set forth in any of SEQ ID NO:26, SEQ ID NO:136, or SEQ ID        NO:138;    -   (c) contain in operable position within the polypeptide, at        least one of each of six different motif peptide segments in        consecutive order M0, M1, M2, M3, M4 and M5, each motif peptide        segment exhibiting at least about 80% identity to a consensus        sequence specified for the respective motif peptide segment, in        which the consensus sequence for motif peptide segment M0 is set        forth in SEQ ID NO:31, the consensus sequence for motif peptide        segment M1 is set forth in SEQ ID NO:48, the consensus sequence        for motif peptide segment M2 is set forth in SEQ ID NO:53, the        consensus sequence for motif peptide segment M3 is set forth in        SEQ ID NO:62, the consensus sequence for motif peptide segment        M4 is set forth in SEQ ID NO:65, and the consensus sequence for        motif peptide segment M5 is set forth in SEQ ID NO:139;    -   (d) contain in operable linkage within the polypeptide, at least        one of each of three different motif peptide segments M1t, M2t,        and M4t, in consecutive order, wherein each motif peptide        segment exhibits at least about 80% identity to a consensus        sequence specified for the respective motif peptide segment, and        wherein the consensus sequence for motif peptide segment M1t is        set forth at SEQ ID NO:70, the consensus sequence for motif        peptide segment M2t is set forth at SEQ ID NO:87, and the        consensus sequence for motif peptide segment M4t is set forth at        SEQ ID NO:120;    -   (e) contain an amino acid sequence exhibiting from about 195 to        about 386 amino acid identities to the amino acid sequence set        forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID        NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,        SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID        NO:26, SEQ ID NO:136, or SEQ ID NO:138;    -   (f) contain an amino acid sequence exhibiting at least from        about 56 to about 100% identity to the amino acid sequence set        forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID        NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,        SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24;    -   (g) contain an amino acid sequence exhibiting at least from        about 56% to about 100% identity, or any percentage point in        between 56% to 100% to the amino acid sequence set forth in any        of SEQ ID NO:26, SEQ ID NO:136, and SEQ ID NO:138; or    -   (h) are encoded by a polynucleotide segment that hybridizes        under stringent hybridization conditions to one or more of the        nucleotide sequences set forth in any of SEQ ID NO:1, SEQ ID        NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ        ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID        NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:135, or SEQ ID        NO:137, or the complement thereof.

Insect inhibitory compositions are provided comprising theaforementioned recombinant polypeptides along with methods forcontrolling Lepidopteran and/or Hemipteran species using suchrecombinant polypeptides.

Recombinant polynucleotides are provided comprising a nucleotidesequence encoding the aforementioned recombinant polypeptides.Transgenic plant cells, plants, or plant parts comprising suchrecombinant polynucleotides and methods of controlling a Lepidopteranand/or Hemipteran species pest using such transgenic plant cells, plantsor plant parts are also provided.

Processed plant products are provided that comprise a detectable amountof the recombinant polynucleotide. Such processed products include, butare not limited to, plant biomass, oil, meal, animal feed, flour,flakes, bran, lint, hulls, and processed seed.

Methods of making transgenic plants are also provided. Such methodsinclude introducing the recombinant polynucleotide into a plant cell andselecting a transgenic plant that expresses an insect inhibitory amountof the recombinant polypeptide encoded by the recombinantpolynucleotide.

Other embodiments, features, and advantages of the invention will beapparent from the following detailed description, examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the primary structure of proteins exemplified in thisapplication, showing the features and motifs characteristic for each ofthe proteins in this genus. Each protein is depicted by name, SEQ ID NO,structural schematic, and amino acid length (i.e., number of aminoacids), and organized into groups or clusters based on amino acididentity between the various proteins. The proteins within a groupgenerally exhibit at least about 90% amino acid identity. Generally,most proteins in the genus of novel proteins disclosed herein contain,in operable linkage within the polypeptide, at least one of each of sixdifferent signature motifs (M) or motif peptide segments, each motifbeing unique to this genus of proteins. The motifs are referenced hereinand consecutively numbered as M0, M1, M2, M3, M4 and M5. The M0 motif isproximal to the amino terminus of each protein toxin, and the M5 motifis positioned most proximal to the carboxy terminus of each proteintoxin. The M5 motif can also be present in more than one copy in eachprotein. Each motif contains a core amino acid segment unique to thatparticular motif. The presence of any of the referenced motif peptidesegments in a protein derived from Bacillus thuringiensis or relatedspecies of bacilli, and the observation for such protein of toxicactivity directed to one or more species of Hemiptera and/orLepidoptera, is sufficient to provide for classification of such proteinas being within the scope of the present invention, particularly if thefull length of the protein amino acid sequence exhibits at least about47% or greater identity to any of the proteins embodied herein includingproteins as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24. Secondary motifsobserved within each of the major motifs M0-M5 in certain of theproteins of the present invention are referenced herein as M1t, M2t, andM4t. Certain of the proteins also contain proteolytic cleavage sites KK,EH, TF, and FG, the relative positions of each being marked inapplicable proteins represented in FIG. 1 as [1], [2], [3], and [4],respectively (the two letters represent the two amino acid residuesbracketing the cleavage site), are features of proteins within the scopeof this invention.

FIG. 2 is a Venn diagram depicting the activity profile of the proteinsof the present invention. Circle [A] represents Lepidopteran-activeproteins, while Circle [B] represents Hemipteran-active proteins. Area[‘c’] is an intersection of Circles [A] and [B], which representsproteins that tested positive for activity against both Lepidopteran andHemipteran insects. Area [‘a’] is [A] minus [‘c’], which representsproteins that tested positive for activity against Lepidopteran insectsbut not positive for any activity against Hemipteran species indicatedat the dose at which Lepidopteran activity was observed. Area [‘b’] is[B] minus [‘c’], which represents proteins that tested positive foractivity against Hemipteran insects but not positive for activityagainst the Lepidopteran species indicated at the dose at whichHemipteran species activity was observed.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bacillus thuringiensis (Bt) species havingan open reading frame at nucleotide positions 1-1107 encoding a TIC1498protein.

SEQ ID NO:2 is an amino acid sequence of a TIC1498 protein toxin.

SEQ ID NO:3 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1158 encoding a TIC1415 protein.

SEQ ID NO:4 is an amino acid sequence of a TIC1415 protein toxin.

SEQ ID NO:5 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1158 encoding a TIC1497 protein.

SEQ ID NO:6 is an amino acid sequence of a TIC1497 protein toxin.

SEQ ID NO:7 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1056 encoding a TIC1886 protein.

SEQ ID NO:8 is an amino acid sequence of a TIC1886 protein toxin.

SEQ ID NO:9 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1158 encoding a TIC1925 protein.

SEQ ID NO:10 is an amino acid sequence of a TIC1925 protein toxin.

SEQ ID NO:11 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1053 encoding a TIC1414 protein.

SEQ ID NO:12 is an amino acid sequence of a TIC1414 protein toxin.

SEQ ID NO:13 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1104 encoding a TIC1885 protein.

SEQ ID NO:14 is an amino acid sequence of a TIC1885 protein toxin.

SEQ ID NO:15 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1155 encoding a TIC1922 protein.

SEQ ID NO:16 is an amino acid sequence of a TIC1922 protein toxin.

SEQ ID NO:17 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1056 encoding a TIC1422 protein.

SEQ ID NO:18 is an amino acid sequence of a TIC1422 protein toxin.

SEQ ID NO:19 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1056 encoding a TIC1974 protein.

SEQ ID NO:20 is an amino acid sequence of a TIC1974 protein toxin.

SEQ ID NO:21 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1155 encoding a TIC2032 protein.

SEQ ID NO:22 is an amino acid sequence of a TIC2032 protein toxin.

SEQ ID NO:23 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1104 encoding a TIC2120 protein.

SEQ ID NO:24 is an amino acid sequence of a TIC2120 protein toxin.

SEQ ID NO:25 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bt species having an open reading frame atnucleotide positions 1-1053 encoding a TIC1362 protein.

SEQ ID NO:26 is an amino acid sequence of a TIC1362 protein toxin.

SEQ ID NO:27 is an artificial nucleotide sequence encoding a TIC1415protein.

SEQ ID NO:28 is an artificial nucleotide sequence encoding a TC1414protein.

SEQ ID NO:29 is an artificial nucleotide sequence encoding a TIC1422protein.

SEQ ID NO:30 is an artificial nucleotide sequence encoding a TIC1362protein.

SEQ ID NO:31 is a consensus amino acid sequence for the M0 motifsegment.

SEQ ID NOs:32-47 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:31.

SEQ ID NO:48 is a consensus amino acid sequence for the M1 motifsegment.

SEQ ID NOs:49-52 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:48.

SEQ ID NO:53 is a consensus amino acid sequence for the M2 motifsegment.

SEQ ID NOs:54-61 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:53.

SEQ ID NO:62 is a consensus amino acid sequence for the M3 motifsegment.

SEQ ID NOs:63-64 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:62.

SEQ ID NO:65 is a consensus amino acid sequence for the M4 motifsegment.

SEQ ID NOs:66-69 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:65.

SEQ ID NO:70 is a consensus amino acid sequence for the M1t motifsegment.

SEQ ID NOs:71-86 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:70.

SEQ ID NO:87 is a consensus amino acid sequence for the M2t motifsegment.

SEQ ID NOs:88-119 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:87.

SEQ ID NO:120 is a consensus amino acid sequence for the M4t motifsegment.

SEQ ID NOs:121-122 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:120.

SEQ ID NO:123 is an amino acid sequence representing an insectinhibitory fragment of TIC1497 and corresponds to an amino acidtranslation of nucleotide positions 1 through 933 of SEQ ID NO:5.

SEQ ID NO:124 is an amino acid sequence representing an insectinhibitory fragment of TIC1497 and corresponds to an amino acidtranslation of nucleotide positions 1 through 885 of SEQ ID NO:5.

SEQ ID NO:125 is an amino acid sequence representing an insectinhibitory fragment of TIC1497 and corresponds to an amino acidtranslation of nucleotide positions 1 through 939 of SEQ ID NO:5.

SEQ ID NO:126 is an amino acid sequence representing an insectinhibitory fragment of TIC1497 and corresponds to an amino acidtranslation of nucleotide positions 1 through 882 of SEQ ID NO:5.

SEQ ID NO:127 is an oligonucleotide sequence in a primer for hybridizingto the (+) strand of the 5′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1 . . . 29 of SEQ ID NO:3(tic1415 forward primer).

SEQ ID NO:128 is an oligonucleotide sequence in a primer for hybridizingto the (−) strand of the 3′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1131 . . . 1161 of SEQ ID NO:3(tic1415 reverse primer).

SEQ ID NO:129 is an oligonucleotide sequence in a primer for hybridizingto the (+) strand of the 5′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1 . . . 40 of SEQ ID NO:11(tic1414 forward primer).

SEQ ID NO:130 is an oligonucleotide sequence in a primer for hybridizingto the (−) strand of the 3′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1015 . . . 1056 of SEQ ID NO:11(tic1414 reverse primer).

SEQ ID NO:131 is an oligonucleotide sequence in a primer for hybridizingto the (+) strand of the 5′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1 . . . 35 of SEQ ID NO:17(tic1422 forward primer).

SEQ ID NO:132 is an oligonucleotide sequence in a primer for hybridizingto the (−) strand of the 3′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1021-1059 of SEQ ID NO:17(tic1422 reverse primer).

SEQ ID NO:133 is an oligonucleotide sequence in a primer for hybridizingto the (+) strand of the 5′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1 . . . 28 of SEQ ID NO:25(tic1362 forward primer).

SEQ ID NO:134 is an oligonucleotide sequence in a primer for hybridizingto the (−) strand of the 3′ end of DNA encoding a protein of the presentinvention and corresponds to positions 1025-1056 of SEQ ID NO:25(tic1362 reverse primer).

SEQ ID NO:135 is a nucleotide sequence representing a recombinantpolynucleotide derived from a Bacillus thuringiensis (Bt) species havingan open reading frame at nucleotide positions 1-1008 encoding a TIC2335protein.

SEQ ID NO:136 is an amino acid sequence of a TIC2335 protein toxin.

SEQ ID NO:137 is a nucleotide sequence representing a polynucleotidederived from a Bacillus thuringiensis (Bt) species having an openreading frame at nucleotide positions 1-1014 encoding a TIC2334 protein.

SEQ ID NO:138 is an amino acid sequence of a TIC2334 protein toxin.

SEQ ID NO:139 is a consensus amino acid sequence for the M5 motifsegment.

SEQ ID NOs:140-141 are each individual amino acid sequences from each ofthe various toxin proteins disclosed herein which were used informulating the consensus sequence as set forth in SEQ ID NO:139.

SEQ ID NO:142 is an N-terminal consensus sequence shared by proteins ofthe present invention.

SEQ ID NO:143 is a C-terminal consensus sequence shared by proteins ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Bacillus thuringiensis (Bt) proteins are a rich source of diverse toxinproteins; however, many problems exist in the process of identifying newBt toxins. Screening methods that involve morphological typing of Btstrains, (e.g. structural analysis of parasporal inclusion bodies, cellcoat morphology, visible color, and morphology under different growingconditions), do not provide a good correlation with the presence ofnovel toxic proteins. Additionally, screening methods that involvehighly matrixed bioassay processes for identifying proteins with toxicproperties yield inconsistent results. Such processes include but maynot be limited to testing proteins expressed at various stages of Btgrowth and development, testing different Bt protein preparations,testing Bt proteins activated by various proteolytic treatments, testingBt proteins with other ancillary proteins, and testing Bt proteins undervarious induction conditions. Some screening methods rely on structuraland functional design, which require very labor and skill intensiveprocedures to elucidate structure/function relationships, and oftenthese protocols can only be effective when carried out on fullyelucidated toxins. In view of the inherent problems in finding new Bttoxin proteins, screening for genes encoding Bt toxin proteins haschanged due to recent improvements in bioinformatics and genome sequencecapabilities.

The inventors herein have taken advantage of high throughput sequencingand improvements in bioinformatics capabilities to screen Bt genomes fornovel protein-encoding Bt toxin genes, which are then cloned andexpressed in acrystalliferous Bt strains to produce protein samples forinsect inhibitory activity screening. As described herein and using thismethod, a novel protein genus has been discovered and exemplary proteinsexhibiting insecticidal activity against Hemipteran and/or Lepidopteranspecies. Those skilled in the art will appreciate that the teaching ofthe present invention enables related gene/protein members to beidentified or engineered that exhibit the properties and features of theproteins of the present invention.

The polypeptides/proteins of the present invention are related by sourceor origin (from B.t. strains of bacteria), by biological toxin activityagainst insect pests within the orders Hemiptera and/or Lepidoptera, byprimary structure (conserved amino acid sequences), and by length (fromabout 300 to about 400 amino acids).

Proteins of the present invention, and proteins that resemble theproteins of the present invention, can be identified by comparison toeach other using various computer based algorithms known in the art.Amino acid identities reported herein are a result of a Clustal Walignment using these default parameters: Weight matrix: blosum, Gapopening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps:On, Hydrophilic residues: GPSNDQERK, Residue-specific gap penalties: On(Thompson et al (1994) Nucleic Acids Research, 22:4673-4680).

It is intended that a recombinant polypeptide exhibiting insectinhibitory activity against a Lepidopteran and/or Hemipteran insectspecies is within the scope of the present invention if an alignment ofthe polypeptide with any of SEQ ID NO:2 (TIC1498), SEQ ID NO:4(TIC1415), SEQ ID NO:6 (TIC1497), SEQ ID NO:8 (TIC1886), SEQ ID NO:10(TIC1925), SEQ ID NO:12 (TIC1414), SEQ ID NO:14 (TIC1885), SEQ ID NO:16(TIC1922), SEQ ID NO:18 (TIC1422), SEQ ID NO:20 (TIC1974), SEQ ID NO:22(TIC2032), SEQ ID NO:24 (TIC2120), SEQ ID NO:26 (TIC1362), SEQ ID NO:136(TIC2335), and SEQ ID NO:138 (TIC2334) results in at least 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, or 386 amino acid identities. SeeTable 1. That is, in certain embodiments, the recombinant polypeptide ofthe present invention comprises an amino acid sequence exhibiting195-386 amino acid identities when compared to SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:136, and SEQ ID NO:138.

TABLE 1 Pair-wise matrix display of toxin proteins of the presentinvention.

(M) is SEQ ID NO and protein name (TIC#) (N) is SEQ ID NO. The percentamino acid identity between all pairs is calculated relative to (M) andis represented by the upper numbers. The lower number in each box in thematrix represents the numbers of identical amino acids between the pair.The gray shaded boxes represent closely related toxin proteins which aremore distantly related to the other proteins in the table.

It is also intended that a first protein exhibiting insect inhibitoryactivity is within the scope of the present invention if a Clustal Walignment of such protein with any of the following second proteins setforth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, results in at least about 47% aminoacid sequence identity between the first and the second proteins; orspecifically, at least 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.2, 99.5, 99.8, or 100% amino acid sequenceidentity between the first and the second proteins; or optionally afirst protein exhibiting insect inhibitory activity is within the scopeof the present invention if a Clustal W alignment of such protein withany of the following second proteins set forth in any of SEQ ID NO:26,SEQ ID NO:136, or SEQ ID NO:138, results in at least about 56% aminoacid sequence identity between the first and the second proteins; orspecifically, at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.5,99.8, or 100% amino acid sequence identity between the first and thesecond proteins.

Polypeptides/proteins of the present invention are observed to berelated by the presence of six signature amino acid sequence motifsegments known to exist only in members of this particular insectinhibitory protein family. The relative position of each of thesignature motif segments is illustrated in FIG. 1 as “M0”, “M1”, “M2”,“M3”, “M4”, and “M5”. SEQ ID NO:31 represents the M0 motif consensussequence, in which X₁ is N or T, X₂ is D or A, X₃ is I or T, and X₄ is Ror S. Each M0 motif segment is represented by the corresponding aminoacid sequences set forth in SEQ ID NOs:32-47. The M0 motif segmentcorresponds to amino acid sequence positions 48 through 70 of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:18,and SEQ ID NO:20, positions 47 through 69 of SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24. A core amino acid sequenceQX₂FQTX₃PX₄L (SEQ ID NO:144) is embedded within the M0 motif segment.The presence of this core sequence (or the M0 motif segment), or of apeptide segment exhibiting at least about 80% amino acid sequenceidentity to this core sequence (or the M0 motif segment) in a particulartoxin protein derived from Bt, alone or in combination with other motifsegments described herein and operably positioned within the primarysequence of any such toxin protein, is determinative that the toxinprotein is a member of the genus of proteins described herein,particularly when the protein is also shown to exhibit insect inhibitoryproperties.

SEQ ID NO:48 represents the M1 motif consensus sequence, in which X₁ isV or I, and X₂ is R or K. Each M1 motif is represented by thecorresponding amino acid sequences set forth in SEQ ID NOs:49-52. The M1motif corresponds to amino acid sequence positions 76 through 118 of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:18, and SEQ ID NO:20, and positions 75 through 117 of SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24, or anyshorter segment comprising the core amino acid sequence QTX₁SFNEX₂TT(SEQ ID NO:145) of this M1 motif. The presence of this core sequence (orthe M1 motif), or of a peptide segment exhibiting at least 80% aminoacid sequence identity to this core sequence (or the M1 motif), in aparticular protein derived from Bt, alone or in combination with othermotifs described herein, is determinative that the protein is a memberof the genus of proteins described herein, particularly when the proteinis also shown to exhibit insect inhibitory properties. Certain proteinswithin the genus of proteins exemplified herein include the M1 motif aswell as a secondary core motif segment Mt1 represented by the consensusamino acid sequence as set forth in SEQ ID NO:70, in which X₁ is V or F,X₂ is S or T, X₃ is H or T, and X₄ is V or T. Each M1t secondary coremotif segment is represented by the amino acid sequences set forth inSEQ ID NOs:71-86. M1t corresponds to amino acid positions 94 through 112of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:18, and SEQ ID NO:20, positions 93 through 111 of SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24, andpositions 83 through 101 of SEQ ID NO:26. The presence of this secondarycore motif M1t, or of a peptide segment exhibiting at least 80% aminoacid sequence identity to this secondary core motif, in a particularprotein derived from Bt, alone or in combination with other motifsdescribed herein, is determinative that the protein is a member of thegenus of proteins described herein, particularly when the protein isalso shown to exhibit insect inhibitory properties.

SEQ ID NO:53 represents the M2 motif consensus sequence, in which X₁ isS or A, X₂ is V or T, and X₃ is T or S. Each M2 motif is represented bythe corresponding amino acid sequences set forth in SEQ ID NOs:54-61.The M2 motif corresponds to amino acid positions 134 through 170 of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:18, and SEQ ID NO:20, and positions 133 through 169 of SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24. The presenceof this M2 motif, or of a peptide segment exhibiting at least 80% aminoacid sequence identity to this M2 motif, in a particular protein derivedfrom Bt, alone or in combination with other motifs described herein, isdeterminative that the protein is a member of the genus of proteinsdescribed herein, particularly when the protein is also shown to exhibitinsect inhibitory properties. Certain proteins within the genus ofproteins exemplified herein include as a part of the M2 motif asecondary motif M2t as set forth in SEQ ID NO:87, in which X₁ is E or A,X₂ is G or S, X₃ is V or T, X₄ is T or S, X₅ is L or I. Each M2t motifis represented by amino acid sequences set forth in SEQ ID NOs:88-119.M2t corresponds to amino acid positions 153 through 168 of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:18, andSEQ ID NO:20, positions 152 through 167 of SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24, and positions 139 through154 of SEQ ID NO:26. The presence of this motif M2t or of a peptidesegment exhibiting at least 80% amino acid sequence identity to thismotif M2t in a particular protein derived from Bt, alone or incombination with other motifs described herein, is determinative thatthe protein is a member of the genus of proteins described herein,particularly when the protein is also shown to exhibit insect inhibitoryproperties.

SEQ ID NO:62 represents the M3 motif consensus sequence, in which X₁ isD or N. Each M3 motif is represented by amino acid sequences set forthin SEQ ID NOs:63-64. M3 corresponds to amino acid positions 172 through200 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:18, and SEQ ID NO:20, and positions 171 through 199 of SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:22, and SEQ ID NO:24, orany shorter segment comprising the core amino acid sequence AGSVX₁VPID(SEQ ID NO:146) of this M3 motif. The presence of this M3 motif or itscore, or of a peptide segment exhibiting at least 80% amino acidsequence identity to this M3 motif or to its core sequence, alone or incombination with other motifs described herein, in a particular proteinderived from Bt is determinative that the protein is a member of thegenus of proteins described herein, particularly when the protein isalso shown to exhibit insect inhibitory properties.

SEQ ID NO:65 represents the M4 motif consensus sequence, in which X₁ isP or T, and X₂ is D or N. Each M4 motif is represented by amino acidsequences set forth in SEQ ID NOs:66-69. M4 corresponds to amino acidpositions 267 through 294 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:18, and SEQ ID NO:20, and positions 266through 293 of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ IDNO:22, or any shorter segment comprising the core amino acid sequenceSLATX₁X₂QILS (SEQ ID NO:147) of this M4 motif. The presence of this M4motif or its core, or of a peptide segment exhibiting at least 80% aminoacid sequence identity to this M4 motif or to its core sequence, aloneor in combination with other motifs described herein, in a particularprotein derived from Bt, is determinative that the protein is a memberof the genus of proteins described herein, particularly when the proteinis also shown to exhibit insect inhibitory properties. Certain proteinswithin the genus of proteins exemplified herein include as a part of theM4 motif a secondary motif M4t as set forth in SEQ ID NO:120, in whichX₁ is A or T. Each M4t motif is represented by amino acid sequences SEQID NO:121 and SEQ ID NO:122. The M4t motif corresponds to amino acidpositions 267 through 281 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:18, and SEQ ID NO:20, positions 266through 280 of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ IDNO:22, and positions 254 through 268 of SEQ ID NO:26, or any shortersegment comprising the core amino acid sequence PGFTGETR (SEQ IDNO:148). The presence of this M4t motif, or of a peptide segmentexhibiting at least 80% amino acid sequence identity to this M4t motif,alone or in combination with other motifs described herein, in aparticular protein derived from Bt, is determinative that the protein isa member of the genus of proteins described herein, particularly whenthe protein is also shown to exhibit insect inhibitory properties.

SEQ ID NO:139 represents the M5 motif consensus sequence segment, inwhich X₁ is C, Y or R, X2 is H or R, X3 is N, D or H, X4 is Y or H, X5is R or G, and X6 is D or N. SEQ ID NOs:142-143 are two exemplary M5motifs. M5 corresponds to amino acid positions 327 through 343 of SEQ IDNOs: 12, 14, 16, 22, and 24, positions 328 through 344 of SEQ ID NOs: 2,4, 6, 8, 10, 18, and 20, positions 344 through 360 of SEQ ID NOs: 14,16, 22, and 24, positions 345 through 361 of SEQ ID NOs: 2, 4, 6, and10, positions 361 through 377 of SEQ ID NOs: 12, 16, and 22, positions362 through 378 of SEQ ID NOs: 4, 6, and 10, or any shorter segmentcomprising the core amino acid sequence (C/Y)EHNYDE (SEQ ID NO:149) ofthis M5 motif. The core amino acid sequence (C/Y)EHNYDE (SEQ ID NO:149)of this M5 motif corresponds to seven of the last 8 N-terminal aminoacid residues in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and24. The presence of this M5 motif or its core, or of a peptide segmentexhibiting at least 80% amino acid sequence identity to this M5 motif,alone or in combination with other motifs described herein, in aparticular protein derived from Bt is determinative that the protein isa member of the genus of proteins described herein, particularly whenthe protein is also shown to exhibit insect inhibitory properties. Thepolypeptides/proteins of the present invention are related by this M5motif which can occur once as exemplified by SEQ ID NOs: 8, 12, 18, and20; as a double repeat as exemplified by SEQ ID NOs: 2, 14, and 24; andas a triple repeat as exemplified by SEQ ID NOs: 4, 6, 10, 12, 16, and22. Interestingly, TIC1414 and TIC1922 can be aligned with 100% identitywith reference to TIC1414 (Table 1), which is possible because thedifference between TIC1414 and TIC1922 are two repeats of the M5 motif(gaps allowed in the pair-wise alignment).

The present invention provides a recombinant polypeptide comprising apeptide segment exhibiting at least 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:31 (motif M0), SEQ ID NO:48 (motif M1), SEQ ID NO:53 (motifM2), SEQ ID NO:62 (motif M3), SEQ ID NO:65 (motif M4), SEQ ID NO:141(motif M5), SEQ ID NO:70 (motif M1t), SEQ ID NO:87 (motif M2t), SEQ IDNO:120 (motif M4t), and any combination thereof. Such polypeptideexhibits insect inhibitory activity against Lepidopteran and/orHemipteran species. As used herein, the term “insect inhibitoryactivity” refers to activity of a protein, or a fragment thereof,effective in inhibiting a pest, preferably a pest of one or more cropplants, when provided in the diet of the pest and ingested by the target(intended) pest. Pests of crop plants include nematodes and arthropods,including insects. Proteins of the present invention are effective ininhibiting the growth, development, viability or fecundity of aparticular target pest, particularly an insect pest, including but notlimited to insects of the orders Lepidoptera and Hemiptera.

In certain embodiments, the insect inhibitory polypeptides/proteinscontain amino acid segments exhibiting at least 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identityto any one or more of the signature motifs (SEQ ID NOs:31-122, 142 and143) of the proteins of the present invention.

In certain embodiments, the recombinant polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:136, or SEQ ID NO:138, or an insect inhibitory fragment thereof.Exemplary insect inhibitory fragments include, but not limited to, thosecomprising the amino acid sequence as set forth in SEQ ID NO:123, SEQ IDNO:124, SEQ ID NO:125, and SEQ ID NO:126.

Additional signature motifs include proteolytic cleavage sites “KK”,“EH”, “TF”, and “FG”, the relative positions of each shown as [1], [2],[3], and [4], respectively in FIG. 1.

An additional signature motif includes an N-terminal consensus sequenceas set forth in SEQ ID NO:142, where X₁ is A or E, X₂ is N or D, X₃ is Qor E, and X₄ is S or L, shared by proteins that are members of the genusof proteins exemplified herein, with the exception of the protein havingthe amino acid sequence as set forth in SEQ ID NO:26. Forwardoligonucleotide primers, e.g., SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, and SEQ ID NO:133 can be designed to hybridize to the plusstrand of the DNA sequence encoding for the N-terminal consensussequence of proteins of the present invention. The C-terminal consensussequence as set forth in SEQ ID NO:143, where X₁ is H or E, and X₂ is Nor Y, is a signature motif shared by proteins of the present invention.Reverse oligonucleotide primers, e.g., SEQ ID NO:128, SEQ ID NO:130, SEQID NO:132, and SEQ ID NO:134, can be designed to hybridize to the minusstrand of the DNA sequence encoding for the C-terminus consensussequence of proteins of the present invention. Oligonucleotide primerscan be designed to hybridize to plus or minus strands of any one or moreof the signature motifs (SEQ ID NOs:31-122, 140 and 141) of the proteinsof the present invention.

When combined, forward and reserve primers can be used to amplifynucleotide sequences encoding proteins (or fragments thereof) of thepresent invention.

Using a Venn diagram (FIG. 2) together with bioassay evidencedemonstrating toxin activity, the relationships of the proteins of thepresent invention are illustrated by common function and insecticidalactivity towards Hemipteran and/or Lepidopteran insect species. Table 2correlates the proteins illustrated in the Venn diagram of FIG. 2 toinsect inhibitory activity by insect species. The results from whichTable 2 was assembled are described in more detail in the examples.

TABLE 2 Activity profiles of exemplary proteins of the present inventionInsect Insect [‘a’] [‘c’] [‘b’] Order Species TIC1422 TIC1886 TIC1498TIC1497 TIC1415 TIC1414 TIC1362 TIC1922 TIC1974 Lepidoptera H. zea M O.nubilalis M/S M/S M/S S D. saccharalis M M/S M/S M/S D. grandiosella MM/S M/S M A. gemmatalis M/S M/S M/S M/S M/S Hemiptera L. lineolaris MM/S M/S* M/S M M M/S S L. Hesperus M M/S M/S* M M/S M = observedMortality (compared to buffer control) S = observed Stunting ofsurvivors (compared to buffer control) *= N-terminal segment that hasbeen truncated at its C-terminus also demonstrated insect inhibitoryactivity [‘a’], [‘b’], and [‘c’] are Venn diagram regions depicted inFIG. 2.

In certain embodiments, the pest is specifically an insect pest. Insectpests include insects selected from the orders Coleoptera, Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Blattodea,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,and Trichoptera.

The insects can include larvae of the order Lepidoptera, such as but notlimited to, armyworms, cutworms, loopers, and heliothines in the FamilyNoctuidae (e.g. fall armyworm (Spodoptera frugiperda), beet armyworm(Spodoptera exigua), bertha armyworm (Mamestra configurata), blackcutworm (Agrotis ipsilon), cabbage looper (Trichoplusia ni), soybeanlooper (Pseudoplusia includens), velvetbean caterpillar (Anticarsiagemmatalis), green cloverworm (Hypena scabra), tobacco budworm(Heliothis virescens), granulate cutworm (Agrotis subterranea), armyworm(Pseudaletia unipuncta), western cutworm (Agrotis orthogonia); borers,casebearers, webworms, coneworms, cabbageworms and skeletonizers fromthe Family Pyralidae (e.g., European corn borer (Ostrinia nubilalis),navel orangeworm (Amyelois transitella), corn root webworm (Crambuscaliginosellus), sod webworm (Herpetogramma licarsisalis), sunflowermoth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae (e.g. codling moth (Cydia pomonella), grape berrymoth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidopteran insects (e.g., diamondback moth (Plutellaxylostella), pink bollworm (Pectinophora gossypiella), gypsy moth(Lymantria dispar). Other insect pests of order Lepidoptera include,e.g., Alabama argillacea (cotton leaf worm), Archips argyrospila (fruittree leaf roller), A. rosana (European leaf roller) and other Archipsspecies, Chilo suppressalis (Asiatic rice borer, or rice stem borer),Cnaphalocrocis medinalis (rice leaf roller), Crambus caliginosellus(corn root webworm), C. teterrellus (bluegrass webworm), Diatraeagrandiosella (southwestern corn borer), D. saccharalis (surgarcaneborer), Earias insulana (spiny bollworm), E. vittella (spottedbollworm), Helicoverpa armigera (American bollworm), H. zea (cornearworm or cotton bollworm), Heliothis virescens (tobacco budworm),Herpetogramma licarsisalis (sod webworm), Lobesia botrana (Europeangrape vine moth), Pectinophora gossypiella (pink bollworm),Phyllocnistis citrella (citrus leafminer), Pieris brassicae (large whitebutterfly), P. rapae (imported cabbageworm, or small white butterfly),Plutella xylostella (diamondback moth), Spodoptera exigua (beetarmyworm), S. litura (tobacco cutworm, cluster caterpillar), S.frugiperda (fall armyworm), and Tuta absoluta (tomato leafminer).

The insects can include adults and nymphs of the orders Hemiptera andHomoptera, such as but not limited to, plant bugs from the FamilyMiridae, cicadas from the Family Cicadidae, leafhoppers (e.g., Empoascaspp.) from the Family Cicadellidae, planthoppers from the familiesFulgoroidea and Delphacidae, treehoppers from the Family Membracidae,psyllids from the Family Psyllidae, whiteflies from the FamilyAleyrodidae, aphids from the Family Aphididae, phylloxera from theFamily Phylloxeridae, mealybugs from the Family Pseudococcidae, scalesfrom the families Coccidae, Diaspididae and Margarodidae, lace bugs fromthe Family Tingidae, stink bugs from the Family Pentatomidae, cinch bugs(e.g., Blissus spp.) and other seed bugs from the Family Lygaeidae,spittlebugs from the Family Cercopidae squash bugs from the FamilyCoreidae, and red bugs and cotton stainers from the FamilyPyrrhocoridae. Other pests from the order Hemiptera include Acrosternumhilare (green stink bug), Anasa tristis (squash bug), Blissusleucopterus leucopterus (chinch bug), Corythuca gossypii (cotton lacebug), Cyrtopeltis modesta (tomato bug), Dysdercus suturellus (cottonstainer), Euschistus servus (brown stink bug), Euschistus variolarius(one-spotted stink bug), Graptostethus spp. (complex of seed bugs),Leptoglossus corculus (leaf-footed pine seed bug), Lygus lineolaris(tarnished plant bug), Lygus hesperus (Western tarnish plant bug),Nezara viridula (southern green stink bug), Oebalus pugnax (rice stinkbug), Oncopeltus fasciatus (large milkweed bug), and Pseudatomoscelisseriatus (cotton fleahopper).

In certain embodiments, the recombinant polypeptide of the presentinvention exhibits insect inhibitory activity against Lepidopteranspecies selected from the group consisting of H. zea, O. nubilalis, D.saccharalis, D. grandiosella, A. gemmatalis, S. frugiperda, S. exigua,A. ipsilon, T. ni, P. includens, H virescens, P. xylostella, P.gossypiella, H armigera, E. lignosellus, and P. citrella, and/or againstHemipteran species selected from the group consisting of L. hesperus, L.lineolaris, A. hilare, E. servus, N. viridula, M. persicae, A. glycines,and A. gossypii.

The proteins of the present invention represent a new category and classof Cry protein, exhibiting no greater than 56% amino acid identity toany other Bt protein known in the art. The protein exhibiting thenearest identity to any of the proteins of the present invention isCry15Aa1 (GI: 142726, ACCESSION: AAA22333) (Brown and Whiteley, Journalof Bacteriology, January 1992, p. 549-557, Vol. 174, No. 2). Cry15Aa1was aligned using Clustal W to each protein exemplified in the presentinvention and the results are shown in Table 3.

TABLE 3 Alignment of proteins to Cry15Aa1. Amino acid Percent aminoidentities* acid identity* Protein with Cry15Aa1 with Cry15Aa1 TIC1498(SEQ ID NO: 2) 159 43.1% TIC1415 (SEQ ID NO: 4) 162 42.0% TIC1497 (SEQID NO: 6) 164 42.5% TIC1886 (SEQ ID NO: 8) 163 46.3% TIC1925 (SEQ ID NO:10) 164 42.5% TIC1414 (SEQ ID NO: 12) 159 45.3% TIC1885 (SEQ ID NO: 14)159 43.2% TIC1922 (SEQ ID NO: 16) 160 41.6% TIC1422 (SEQ ID NO: 18) 15644.3% TIC1974 (SEQ ID NO: 20) 156 44.3% TIC2032 (SEQ ID NO: 22) 15540.8% TIC2120 (SEQ ID NO: 24) 158 42.9% TIC1362 (SEQ ID NO: 26) 19455.3% TIC2335 (SEQ ID NO: 136) 130 38.7% TIC2334 (SEQ ID NO: 138) 12938.2% *in a Clustal W alignment

Cry15Aa1 does not contain any of the signature motifs (SEQ IDNOs:31-122, 140 and 141) shared by the proteins of the presentinvention. Cry15Aa1 does not exhibit the proteolytic cleavage sites [2],[3], and [4] shared by the proteins of the present invention as shown inFIG. 1. Cry15Aa1 exhibits a calculated isoelectric point of about 7.3pI, in contrast to the proteins of the present invention which eachexhibits a calculated isoelectric point of about 5 to 6 pI. Cry15Aa1exhibits only 3 positive charges at neutral pH, whereas the proteins ofthe present invention exhibit calculated from 3 to 10 negative chargesat neutral pH.

The proteins of the present invention can be used to produce antibodiesthat bind specifically to this genus of proteins and can be used toscreen for and to find other members of the genus.

Nucleotide sequences encoding these proteins can be used as probes andprimers for screening to identify other members of the genus usingthermal or isothermal amplification and/or hybridization methods, e.g.,oligonucleotides as set forth in SEQ ID NOs:127-134, andoligonucleotides hybridizing to sequence encoding the signature motifsof the present invention. Nucleotide sequence homologs, i.e.,insecticidal proteins encoded by nucleotide sequences that hybridize toeach or any of the sequences disclosed herein under stringenthybridization conditions, are specifically intended to be includedwithin the scope of the present invention. The present invention alsoprovides a method for detecting a first nucleotide sequence thathybridizes to a second nucleotide sequence, wherein the first nucleotidesequence encodes an insecticidal protein or insecticidal fragmentthereof and hybridizes under stringent hybridization conditions to thesecond nucleotide sequence. In such case the second nucleotide sequencecan be any of the sequences disclosed herein under stringenthybridization conditions. Nucleotide coding sequences hybridize to oneanother under appropriate hybridization conditions and the proteinsencoded by these nucleotide sequences cross react with antiserum raisedagainst any one of the other proteins. Stringent hybridizationconditions, as defined herein, comprise at least hybridization at 42° C.followed by two washes for five minutes each at room temperature with2×SSC, 0.1% SDS, followed by two washes for thirty minutes each at 65°C. in 0.5×SSC, 0.1% SDS. Of course, one skilled in the art willrecognize that, due to the redundancy of the genetic code, many othersequences are capable of encoding such related proteins, and thosesequences, to the extent that they function to express insecticidalproteins either in Bacillus strains or in plant cells, are intended tobe encompassed by the present invention, recognizing of course that manysuch redundant coding sequences will not hybridize under theseconditions to the native Bt sequences encoding TIC1498, TIC1415,TIC1497, TIC1886, TIC1925, TIC1414, TIC1885, TIC1922, TIC1422, TIC1974,TIC2032, TIC2120, TIC1362, TIC2335, and TIC2334.

In certain embodiments, a recombinant polypeptide exhibiting insectinhibitory activity against a Lepidopteran and/or Hemipteran insectspecies is within the scope of the present invention, which polypeptideis encoded by a polynucleotide segment that hybridizes under stringenthybridization conditions to one or more of the nucleotide sequences setforth in any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:135, or SEQID NO:137, or the complement thereof.

An aspect of this invention provides methods for discovering relatedproteins, and such methods include the sequencing of Bt genomes,assembly of sequence data, the identification and cloning of Bt genesencoding such insect inhibitory proteins, and the expression and testingof new Bt proteins to assay for insect inhibitory activity. Anotheraspect of this invention employs molecular methods to engineer and clonecommercially useful proteins comprising chimeras of proteins andimproved variants from the genus of insect inhibitory proteins, e.g.,the chimeras can be assembled from segments in each of the variousproteins that are within the spaces between the signature motifs toderive improved embodiments. The proteins of the present invention canbe subjected to alignment to each other and to other Bt insectinhibitory proteins, and segments of each such protein can be identifiedthat may be useful for substitution between the aligned proteins,resulting in the construction of chimeric proteins. Such chimericproteins can be subjected to pest bioassay analysis and characterizedfor the presence of increased bioactivity or expanded target pestspectrum compared to the parent proteins from which each such segment inthe chimera was derived. The insect inhibitory activity of thepolypeptides can be further engineered for improved activity to aparticular pest or to a broader spectrum of pests by swapping domains orsegments with other proteins.

One skilled artisan understands the concept of amino acid substitution,and recognizes that this requires experimentation that is not routine,as there are amino acid positions that can accept substitution withoutapparent affect to the structure or function of the protein; however, insurprising circumstances, even a conservative substitution may bedetermined to significantly alter the structure or function of theprotein, and it is often unknown with precision the positions in theamino acid segments that would accept such changes. Accordingly, aminoacid substitutions at positions along the length of the protein sequencethat affect function can be identified by alanine scanning mutagenesis,and such positions can often be useful for points of amino acidinsertions and/or deletions, or N- or C-terminal deletions. Accordingly,the proteins of the present invention include functionally equivalentfragments (N- or C-terminal deletions) of the proteins represented bythe amino acid sequences of the present invention. N-terminal proteinfragments (SEQ ID. NOs:123-126, 16) of TIC1497 and TIC1922 havedemonstrated insect inhibitory activity (Table 2 and Examples 6, 10, and11, respectively). Corresponding N-terminal protein fragments for anymember of the genus is contemplated.

Proteins functionally equivalent (having substantially equivalent insectinhibitory activity) to the proteins of the present invention includeproteins with conservative amino acid substitutions in the proteinsequences of the present invention. In such amino acid sequences, one ormore amino acids in the starting sequence is (are) substituted withanother amino acid(s), the charge and polarity of which is similar tothat of the native amino acid, i.e., as exemplified herein aconservative amino acid substitution, resulting in a conservative changefrom the perspective of charge and polarity, but which may result in achange in the bioactivity of the protein, preferably increasing theactivity of the protein compared to the starting protein with theoriginal amino acid at such positions, or resulting in a change in thevariant protein with reference to the spectrum of biological activityand without any loss of insect inhibitory activity. An example ofproteins that can entertain substituted amino acids or terminaldeletions to obtain biological equivalents include, but are not limitedto, the protein sequence as set forth in any of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, and SEQ ID NOs:123-126.

Enrichment of the proteins of the present invention either in plants orby a process that includes culturing recombinant Bt cells underconditions to express/produce recombinant polypeptide/proteins of thepresent invention is contemplated. Such a process can includepreparation by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof recombinant Bacillus thuringiensis cells expressing/producing saidrecombinant polypeptide. Such a process can result in a Bt cell extract,cell suspension, cell homogenate, cell lysate, cell supernatant, cellfiltrate, or cell pellet. By obtaining the recombinantpolypeptides/proteins so produced, a composition that includes therecombinant polypeptides/proteins can include bacterial cells, bacterialspores, and parasporal inclusion bodies and can be formulated forvarious uses, including agricultural insect inhibitory spray products oras insect inhibitory formulations in diet bioassays.

It is intended that an insect inhibitory composition/formulationcomprising the aforementioned recombinant polypeptide/protein is withinthe scope of the present invention. In certain embodiments, suchcomposition may further comprise at least one pesticidal agent thatexhibits insect inhibitory activity against the same Lepidopteran and/orHemipteran insect species but is different from the recombinantpolypeptide. Such agent is selected from the group consisting of aninsect inhibitory protein, an insect inhibitory dsRNA molecule, and anancillary protein. Examples of such agents include, but are not limitedto, a TIC807 protein, a TIC853 protein, a AXMI-171 protein, and aCry51Aa1 protein. Other compositions are contemplated for combining withthe proteins of the present invention, and with the combinations ofproteins provided above. For example, topically applied pesticidalchemistries that are designed for controlling pests that are alsocontrolled by the proteins of the present invention can be used with theproteins of the present invention in seed treatments, spray on/dripon/or wipe on formulations that can be applied directly to the soil (asoil drench), applied to growing plants expressing the proteins of thepresent invention, or formulated to be applied to seed containing one ormore transgenes encoding one or more of the proteins of the presentinvention. Such formulations for use in seed treatments can be appliedwith various stickers and tackifiers known in the art. Such formulationsmay contain pesticides that are synergistic in mode of action with theproteins of the present invention, meaning that the formulationpesticides act through a different mode of action to control the same orsimilar pests that are controlled by the proteins of the presentinvention, or that such pesticides act to control pests within a broaderhost range, such as lepidopteran or Hemipteran species or other plantpest species such as coleopteran species that are not effectivelycontrolled by the proteins of the present invention.

The aforementioned composition/formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore/crystal preparation, a seed treatment, arecombinant plant cell/plant tissue/seed/plant transformed to expressone or more of the proteins, or bacterium transformed to express one ormore of the proteins. Depending on the level of insect inhibitory orinsecticidal inhibition inherent in the recombinant polypeptide and thelevel of formulation to be applied to a plant or diet assay, thecomposition/formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

The proteins of the invention can be combined in formulations fortopical application to plant surfaces, to the soil, in formulations forseed treatments, in formulations with other agents toxic to the targetpests of Hemipteran and Lepidopteran species. Such agents include butare not limited to, a TIC807 protein, a TIC853 protein, a AXMI-171protein, and a Cry51Aa1 protein which each are effective in controllingthe same Hemipteran pests that are controlled by the insect inhibitoryproteins of the present invention.

It is also intended that a method of controlling a Lepidopteran and/orHemipteran species pest is within the scope of the present invention.Such method comprises the steps of contacting the pest with an insectinhibitory amount of the recombinant polypeptide/protein. In certainembodiments, Lepidopteran and Hemipteran species pest is in a cropfield.

An embodiment of the invention includes recombinant polynucleotides thatencode the insect inhibitory protein members of the genus. Withreference to a “recombinant” polynucleotide, it is intended that apolynucleotide molecule is made by human means or intervention throughmolecular biology engineering techniques, which can include theamplification or replication of such molecules upon introduction into ahost cell, and the subsequent extraction and/or purification of thepolynucleotide from the representative host cell. Polynucleotideembodiments of the present invention include ribonucleic acids (RNA) anddeoxyribonucleic acids (DNA). Proteins of the present invention can beexpressed from DNA constructs in which the open reading frame encodingthe protein is operably linked to elements such as a promoter and anyother regulatory elements functional for expression in that particularsystem for which the construct is intended. For example,plant-expressible promoters can be operably linked to protein encodingsequences for expression of the protein in plants, and Bt-expressiblepromoters can be operably linked to the protein encoding sequences forexpression of the protein in Bt. Other useful elements that can beoperably linked to the protein encoding sequences include, but are notlimited to, enhancers, introns, protein immobilization tags (HIS-tag),target sites for post-translational modifying enzymes, dsRNA codingsegments, siRNAs, miRNAs, ribosomal binding sites, leader elements, andmiRNA target sites.

Exemplary recombinant polynucleotide molecules provided herewithinclude, but are not limited to, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:135.

An aspect of the invention provides a recombinant DNA construct thatincludes one or more aforementioned polynucleotides, which canadditionally be engineered with transcribable or non-transcribableregions or both. Such regions are operably assembled to promoteexpression of DNA to RNA through either in vivo or in vitro systems,thereby producing the novel RNA transcript embodiments of the presentinvention. The present invention features RNA transcripts that include,but are not limited to, the protein-encoding RNA and additional RNAregions that are translatable, non-translatable, or both. Suchadditional RNA regions include translatable regions engineered totranslate to terminal or intra-peptide regions, and non-translatableregions engineered to either promote transcription, translation, orboth.

In certain embodiments, the aforementioned recombinant DNA construct isin an expression cassette for use in an E. coli or Bt expression system.Expression cassettes are typically designed with a promoter at the 5′end of the cassette, upstream of a desired polynucleotide segmentencoding a protein of the present invention. A promoter can consist ofmultiple different promoter elements operably linked to provide for theinitiation of transcription of the sequences encoding a protein of theinvention. The DNA sequence consisting of the promoter-protein-encodingDNA can be operably linked at its 3′ end to a transcriptionaltermination signal sequence functional in an E. coli and/or Bt cell toproduce the recombinant DNA construct.

In certain embodiments, the aforementioned recombinant DNA construct isin an expression cassette for expression in plants. Expression cassettesare designed with a promoter at the 5′ end of the cassette, upstream ofa desired polynucleotide segment encoding a protein of the presentinvention. 5′ untranscribed DNA can comprise a promoter which canconsist of multiple different promoter and enhancer elements operablylinked to provide for the initiation of transcription of downstreamsequences including sequences encoding the polypeptides of theinvention. One or more transcribed but non-translated DNA sequence(s)can be operably linked 3′ to the promoter in the expression cassette,including leader and/or intron sequence(s). An intron sequence isoptionally provided 3′ to the leader sequence or in some cases withinthe open reading frame encoding the desired protein. A polynucleotidesegment encoding an optional translocation polypeptide (a signal peptideor a chloroplast transit peptide, for example) may be inserted 5′ to thecoding sequence of the protein of the present invention for localizingthe protein of the invention to a particular subcellular position. Thenucleotide sequence encoding the protein of the present invention isoptionally operably positioned within the aforementioned expressioncassette, along with any requisite operably linked polyadenylation(polyA) and/or transcriptional termination sequence functional in plantcells. The aforementioned elements are arranged contiguously and can beused in various combinations depending on the desired expressionoutcome.

The present invention features promoters functional in plants including,but not limited to, constitutive, non-constitutive, spatially-specific,temporally-specific, tissue-specific, developmentally-specific,inducible, and viral promoters. Examples of promoters functional inplants include corn sucrose synthetase 1, corn alcohol dehydrogenase 1,corn light harvesting complex, corn heat shock protein, pea smallsubunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmidnopaline synthase, petunia chalcone isomerase, bean glycine rich protein1, Potato patatin, lectin, CaMV 35S, the S E9 small subunit RuBPcarboxylase, carnation etched ring virus, and dahlia mosaic viruspromoter.

A recombinant DNA construct comprising the protein encoding sequencescan also further comprise a region of DNA that encodes for one or moreinsect inhibitory agents which can be configured to concomitantlyexpress or co-express with DNA sequence encoding the protein of thepresent invention, a protein different from the aforementioned protein,an insect inhibitory dsRNA molecule, or an ancillary protein. Ancillaryproteins include co-factors, enzymes, binding-partners, or other insectinhibitory agents that function synergistically to aid in theeffectiveness of an insect inhibitory agent, for example, by aiding itsexpression, influencing its stability in plants, optimizing free energyfor oligomerization, augmenting its toxicity, and increasing itsspectrum of activity.

A recombinant polynucleotide or recombinant DNA construct comprising theprotein-coding sequence can be delivered to host cells by vectors.Methods for transferring recombinant DNA constructs to and from hostcells, including E. coli, B. thuringiensis, and Agrobacterium species,are known in the art. Such vectors are designed to promote the uptake ofvector DNA and to further provide expression of DNA to RNA to protein inin vitro or in vivo systems, either transiently or stably. Examples ofthe vectors include, but are not limited to, a plasmid, baculovirus,artificial chromosome, virion, cosmid, phagemid, phage, or viral vector.Such vectors can be used to achieve stable or transient expression ofthe protein encoding sequence in a host cell; and, if the case may be,subsequent expression to polypeptide. An exogenous recombinantpolynucleotide or recombinant DNA construct that comprises the proteinencoding sequence and that is introduced into a host cell is alsoreferred to herein as a “transgene”.

Plasmids can be designed to replicate in E. coli or B. thuringiensis, orboth. Such plasmids contain genetic elements that allow for thereplication and maintenance of such plasmids and for the expression oftransgenes, e.g. aforementioned recombinant DNA constructs, in eitherspecies.

Plant transformation vectors can be designed to allow for theAgrobacterium-mediated transfer of a T-DNA, i.e. transferred DNAcomprising aforementioned recombinant DNA constructs. Such planttransformation vectors contain genetic elements that allow for thereplication and maintenance of such plasmid vectors in E. coli and/orAgrobacterium and are essential for transfer of the T-DNA into a plantgenome.

Transgenic host cells comprising recombinant DNA constructs encodingtoxin proteins of the present invention are also contemplated. Atransgenic host cell can be further defined as a prokaryotic host cell,i.e. a bacterial cell, e.g., Bacillus thuringiensis, Bacillus subtilis,Bacillus megaterium, Bacillus cereus, Bacillus laterosperous,Escherichia, Salmonella, Agrobacterium, Pseudomonas, or Rhizobium cell,or a eukaryotic host cell, e.g., a plant cell, and each of these typesof cells is also referred to herein as a microbial cell, a microbe, or amicroorganism.

As used herein a “host cell” means a cell that is transformed ortransfected with exogenous recombinant DNA, e.g. by electroporation orby Agrobacterium-mediated transformation or by bombardment usingmicroparticles coated with recombinant DNA, or by transduction or byplasmid transfer or by other means. A host cell of this invention can bea transformed bacterium, e.g. E. coli host cell or Bt host cell orAgrobacterium host cell, or a plant host cell.

Accordingly, a host cell of can be an originally-transformed plant cellthat exists as a microorganism or as a progeny plant cell that isregenerated into differentiated tissue, e.g., into a transgenic plantwith stably-integrated, non-natural recombinant DNA, or seed or pollenderived from a progeny transgenic plant. As used herein a “transgenicplant” includes a plant, plant part, plant cells or seed whose genomehas been altered by the stable integration of recombinant DNA. Atransgenic plant includes a plant regenerated from anoriginally-transformed plant cell and progeny transgenic plants fromlater generations or crosses of a transformed plant. Accordingly,examples of plant parts are leaf, a branch, a bark, a blade, a pollengrain, a stalk, a cell, a stem, a flower, a sepal, a fruit, a root, or aseed.

Transgenic plants expressing the protein(s) of the invention exhibitpest tolerance. Such plants and its cells include alfalfa, banana,barley, bean, berry, brassica, broccoli, cabbage, cactus, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,clover, coconut, coffee, corn, cotton, cucumber, cucurbit, Douglas fir,eggplant, eucalyptus, flax, fruit, garlic, grape, hops, kapok, leek,legume, lettuce, Loblolly pine, melons, millets, nut, oat, olive, onion,ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine,poplar, potato, pumpkin, Radiata pine, radish, rapeseed, rice,rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean,spinach, squash, strawberry, succulent, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, tree, triticale, turf grass, vegetable, watermelon, andwheat plants and cells.

Nucleotides sequences can be constructed that are useful for expressionof these proteins in plant cells, and such plant cells can beregenerated into transgenic plants that can produce seeds containingsuch nucleotide sequences which can be commercialized, bred togetherwith other transgenic plants expressing different Bt insect inhibitoryproteins or other agents toxic to crop pests.

Plant cells can be transformed by multiple mechanisms that are withinthe skill of the art including but not limited to bacterialtransformation systems such as Agrobacterium or Rhizobacterium,electroporation, ballistic mediated systems, and the like.Microprojectile bombardment methods are illustrated in U.S. Pat. No.5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No.5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No.6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn); U.S. Pat. No.6,153,812 (wheat) and U.S. Pat. No. 6,365,807 (rice) andAgrobacterium-mediated transformation is described in U.S. Pat. No.5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No.5,463,174 (canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No.5,846,797 (cotton); U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No.7,026,528 (wheat) and U.S. Pat. No. 6,329,571 (rice), US PatentApplication Publication 2004/0087030 A1 (cotton), and US PatentApplication Publication 2001/0042257 A1 (sugar beet) and in Arencibia etal. (1998) Transgenic Res. 7:213-222 (sugarcane) and other more recentmethods described in US Patent Application Publications 2009/0138985A1(soybean), 2008/0280361A1 (soybean), 2009/0142837A1 (corn), 2008/0282432(cotton), 2008/0256667 (cotton), 2003/0110531 (wheat), U.S. Pat. No.5,750,871 (canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No.6,365,807 (rice). Transformation of plant material can be practiced intissue culture on a nutrient media, e.g., a mixture of nutrients thatwill allow cells to grow in vitro. Recipient cell targets include, butare not limited to, meristem cells, hypocotyls, calli, immature embryosand gametic cells such as microspores, pollen, sperm and egg cells.Callus may be initiated from tissue sources including, but not limitedto, immature embryos, hypocotyls, seedling apical meristems, microsporesand the like. Cells containing a transgenic nucleus are grown intotransgenic plants.

In addition to direct transformation of a plant material with arecombinant DNA, a transgenic plant cell nucleus can be prepared bycrossing a first plant having cells with a transgenic nucleus withrecombinant DNA with a second plant lacking the transgenic nucleus. Forexample, recombinant DNA can be introduced into a nucleus from a firstplant line that is amenable to transformation to transgenic nucleus incells that are grown into a transgenic plant which can be crossed with asecond plant line to introgress the recombinant DNA into the secondplant line. A transgenic plant with recombinant DNA providing anenhanced trait, e.g., enhanced yield, can be crossed with transgenicplant line having other recombinant DNA that confers another trait, forexample herbicide resistance or pest resistance, to produce progenyplants having recombinant DNA that confers both traits. Typically, insuch breeding for combining traits the transgenic plant donating theadditional trait is a male line and the transgenic plant carrying thebase traits is the female line. The progeny of this cross will segregatesuch that some of the plants will carry the DNA for both parental traitsand some will carry DNA for one parental trait; such plants can beidentified by markers associated with parental recombinant DNA, e.g.,marker identification by analysis for recombinant DNA or, in the casewhere a selectable marker is linked to the recombinant DNA, byapplication of the selecting agent such as a herbicide for use with aherbicide tolerance marker, or by selection for the insect inhibitorytrait. Progeny plants carrying DNA for both parental traits can becrossed back into the female parent line multiple times, for exampleusually 6 to 8 generations, to produce a progeny plant withsubstantially the same genotype as one original transgenic parental linebut for the recombinant DNA of the other transgenic parental line.

In the practice of plant transformation, exogenous DNA is typicallyintroduced into only a small percentage of target plant cells in any onetransformation experiment. Cells of this invention can be directlytested to confirm stable integration of the exogenous DNA by a varietyof well-known DNA detection methods or by a variety of well-knownbioactivity assays that test for insect inhibitory activity (furtherdescribed in the examples section). Marker genes can be used to providean efficient system for identification of those cells that are stablytransformed by receiving and integrating a recombinant DNA molecule intotheir genomes. Preferred marker genes provide selective markers whichconfer resistance to a selective agent, such as an antibiotic or anherbicide. Any of the herbicides to which plants of this invention canbe made resistant can be used as agents for selective markers.Potentially transformed cells are exposed to the selective agent. In thepopulation of surviving cells will be those cells where, generally, theresistance-conferring gene is integrated and expressed at sufficientlevels to permit cell survival. Cells may be tested further to confirmstable integration of the exogenous DNA. Commonly used selective markergenes include those conferring resistance to antibiotics such askanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin(aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides suchas glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA orEPSPS). Examples of such selectable markers are illustrated in U.S. Pat.Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047. Markers whichprovide an ability to visually screen transformants can also beemployed, for example, a gene expressing a colored or fluorescentprotein such as a luciferase or green fluorescent protein (GFP).

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developing plantsregenerated from transformed plant cells can be transferred to plantgrowth mix, and hardened off, for example, in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and 25to 250 microeinsteins m 2 s−1 of light, prior to transfer to agreenhouse or growth chamber for maturation. These growth conditionsvary among plant species and are known to those skilled in the art.Plants are regenerated from about 6 weeks to 10 months after atransformant is identified, depending on the initial tissue, and plantspecies. Plants may be pollinated using conventional plant breedingmethods known to those of skill in the art and seed produced, forexample self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of insect inhibitory activity.

Transgenic plants encoding and expressing one or more of the proteins ofthe present invention are grown to (i) generate transgenic plants havingan enhanced trait as compared to a control plant and (ii) producetransgenic seed and haploid pollen of this invention. Such plants withenhanced traits are identified by selection of transformed plants orprogeny seed for the enhanced trait. For efficiency a selection methodis designed to evaluate multiple transgenic plants (events) comprisingthe recombinant DNA, for example multiple plants from 2 to 20 or moretransgenic events. Transgenic plants grown from transgenic seed providedherein demonstrate improved agronomic traits that contribute toincreased insect inhibitory tolerance or increased harvest yield orother traits that provide increased plant value, including, for example,improved seed or boll quality. Of particular interest are cotton,alfalfa, corn, soy, or sugarcane plants having enhanced insectinhibitory resistance against one or more insects of the ordersLepidoptera and/or Hemiptera. Of particular interest are cotton plantshaving enhanced insect inhibitory resistance against an insect of theorder Hemiptera.

The invention provides methods to produce a plant and harvest a cropfrom seed comprising a recombinant polynucleotide molecule encoding theinsect inhibitory polypeptides of the present invention. Of particularinterest are cotton, alfalfa, corn, soy, or sugarcane plants havingenhanced insect inhibitory resistance against an insect(s) of the orderLepidoptera and/or Hemiptera. The method includes the steps of crossingan insect resistant plant expressing the recombinant polypeptides of thepresent invention with another plant, obtaining at least one progenyplant derived from this cross, and selecting progeny that expresses therecombinant polypeptides of the present invention wherein said progenyis resistant against an insect. This includes the steps of planting theseed, producing a crop from plants grown from the seed, and harvestingthe crop, wherein at least 50% of the crop comprises seed comprising therecombinant polynucleotide molecule.

In an aspect of the invention, a transgenic plant cell, a transgenicplant, and transgenic plant parts comprising a recombinantpolynucleotide (i.e. transgene) that expresses any one or more of theprotein encoding sequences are provided herein. It is intended that“bacterial cell” or “bacterium” can include, but are not limited to, anAgrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas,or a Rhizobium cell. It is intended that “plant cell” or “plant” includean alfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglasfir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce,Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental,palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato,poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye,safflower, shrub, sorghum, Southern pine, soybean, spinach, squash,strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum,sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass,watermelon, and wheat plant cell or plant. In certain embodiments,transgenic plants and transgenic plant parts regenerated from atransgenic plant cell are provided. In certain embodiments, thetransgenic plants can be obtained from a transgenic seed. In certainembodiments, transgenic plant parts can be obtained by cutting,snapping, grinding or otherwise disassociating the part from the plant.In certain embodiments, the plant part can be a seed, a boll, a leaf, aflower, a stem, a root, or any portion thereof. In certain embodiments,a transgenic plant part provided herein is a non-regenerable portion ofa transgenic plant part. As used in this context, a “non-regenerable”portion of a transgenic plant part is a portion that cannot be inducedto form a whole plant or that cannot be induced to form a whole plantthat is capable of sexual and/or asexual reproduction. In certainembodiments, a non-regenerable portion of a plant part is a portion of atransgenic seed, boll, leaf, flower, stem, or root.

Also provided herein are methods of making transgenic plants thatcomprise insect inhibitory amounts of the protein(s) of the presentinvention. Such plants can be made by introducing a recombinantpolynucleotide that encodes any of the proteins provided herein into aplant cell, and selecting a plant derived from said plant cell thatexpresses an insect inhibitory amount of the proteins. Plants can bederived from the plant cells by regeneration, seed, pollen, or meristemtransformation techniques.

Also provided herein is the use of a transgenic plant that expresses aninsect inhibitory amount of one or more of the proteins of the presentinvention to control a Lepidopteran and/or a Hemipteran species pest.Any of the aforementioned transgenic plants can be used in methods forprotecting a plant from insect infestation provided herein.

Also provided herein is the use of any of the aforementioned transgenichost cells to produce the proteins of the present invention.

Additional aspects of the invention include methods and/or kits fordetecting DNA, RNA, or protein of the present invention, methods foridentifying members of the genus of proteins described herein, methodsfor identifying novel proteins related to genus family members, methodsfor testing for control of insect growth and/or infestation, and methodsfor providing such control to plants and other recipient hosts. Theseproteins can be used to produce antibodies that bind specifically tothis class/genus of protein and these antibodies can be used to screenand find other members of the genus. An antibody by itself, or in amixture of antibodies, that binds specifically to a target of therecombinant polypeptides of the present invention is contemplated; and,the method of using this antibody by itself, or in a mixture ofantibodies, to detect or quantify proteins sharing epitopes of theproteins of the present invention is also contemplated. Such a method todetect or quantify can include the steps of contacting a sample with theantibody and using detection means well known in the art to detect thebinding of antibody to polypeptide target in the sample. Where one ormore epitopes are contemplated and their combination used in such amethod, the binding of an antibody or mixture of antibodies recognizingdifferent epitopes can identify a polypeptide exhibiting homology to therecombinant polypeptides of the present invention.

Kits for detecting the presence of a polypeptide target in a samplesuspected of containing the polypeptide target are provided. Such kitswould include a reagent(s) used for epitope detection and a controlreagent(s) to show that the detection was operating within statisticalvariances. Reagent storage, instructions for detection means and use ofreagents, and additional parts and tools that can be included in suchkits are contemplated.

The polynucleotide segments encoding the proteins of the presentinvention, i.e. the proteins of the described genus, particularly thesegments derived from wild type Bt strains, can be used as probes andprimers for screening for and identifying other members within the genususing thermal amplification and/or hybridization methods. Nucleotideprobes or primers can vary in length, sequence, concentration, backbone,and formulation depending on the sample detection method used. Thepresent invention features primers and probes that can be used to detectand isolate homologous genes that encode for insect inhibitory proteinmembers of the genus. A DNA detection kit is contemplated providing askilled artisan to more easily perform the detection and/or isolation ofhomologous genes of the present invention. The invention provides foruse of such kits and methods and for novel genes and the insectinhibitory polypeptides encoded by such genes that are detected andisolated by the aforementioned detection means.

The invention further provides for methods of testing the polypeptidesof the present invention for insect inhibitory activity, herein termed“bioassay”. Described herein are qualitative insect bioassays thatmeasure growth inhibition, mortality, or a combination of both. Theinsect orders tested in the following examples include Coleoptera,Diptera, Lepidoptera, and Hemiptera. The diet recipe and preparation,the preparation of test and control samples, the insect preparation, andthe procedures for conducting assays are typically dependent upon thetype and size of the insect and/or pest being subjected to anyparticular evaluation. Such methods are illustrated and described indetail in the following examples.

In certain embodiments, plant product can comprise commodity or otherproducts of commerce derived from a transgenic plant or transgenic plantpart, where the commodity or other products can be tracked throughcommerce by detecting nucleotide segments or expressed RNA or proteinsthat encode or comprise distinguishing portions of the proteins of thepresent invention. Such commodity or other products of commerce include,but are not limited to, plant parts, biomass, oil, meal, sugar, animalfeed, flour, flakes, bran, lint, processed seed, and seed.

Also provided herewith are processed plant products that comprise adetectable amount of a recombinant nucleotide encoding any one of theproteins of the present invention, an insect inhibitory fragmentthereof, or any distinguishing portion thereof. In certain embodiments,the processed product is selected from the group consisting of plantbiomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, andprocessed seed. In certain embodiments, the processed product isnon-regenerable. In certain embodiments, a distinguishing portionthereof can comprise any polynucleotide encoding at least 20, 30, 50 or100 amino acids of the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 136 or 138.

Also provided herein are methods of controlling insects. Such methodscan comprise growing a plant comprising an insect inhibitory amount ofthe protein of the present invention. In certain embodiments, suchmethods can further comprise any one or more of: (i) applying anycomposition comprising or encoding the proteins of the present inventionto the plant or a seed that gives rise to the plant; and/or (ii)transforming the plant or a plant cell that gives rise to the plant witha polynucleotide encoding the proteins of the present invention. Incertain embodiments, the plant is a transiently or stably transformedtransgenic plant comprising a transgene that expresses an insectinhibitory amount of the protein of the present invention. In certainembodiments, the plant is a non-transgenic plant to which a compositioncomprising the protein of the present invention has been applied.

Other features and advantages of the invention will be apparent from thefollowing detailed description, examples, and claims.

EXAMPLES

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific details disclosed herein arenot to be interpreted as limiting.

Example 1: Discovery of Insect Inhibitory Proteins

Various Bt strains exhibiting distinctive attributes, e.g. inferredtoxicity, proteomic diversity, and morphological variations whencompared to each other, were identified, and DNA was obtained from eachsuch strain and prepared for DNA sequencing. DNA sequence informationwas generated for each such strain, raw sequence reads were processed,contigs were assembled from processed reads, open reading frames wereidentified, and deduced amino acid sequences were analyzed.

Example 2: Cloning and Expressing Insect Inhibitory Proteins

This example illustrates the cloning of polynucleotide segments encodinginsect inhibitory proteins, and insertion into and expression inrecombinant host cells.

Nucleotide segments were obtained by amplification from correspondinggenomic samples from which each open reading frame was identified inExample 1. Amplified nucleotide segments were inserted into arecombinant plasmid and transformed into an acrystalliferous Bt hostcell or into an E. coli expression strain, and the resulting recombinantstrain(s) were observed to express a recombinant protein.

Recombinant proteins exemplified herein were observed to exhibit insectinhibitory properties to a variety of pest species as described inExamples 3-13 below. Nucleotide sequences as set forth in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:135, and SEQ ID NO:137were confirmed to encode proteins having amino acid sequences as setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ IDNO:136, and SEQ ID NO:138 (respectively, TIC1498, TIC1415, TIC1497,TIC1886, TIC1414, TIC1922, TIC1422, TIC1974, TIC1362, TIC2335, andTIC2334).

Recombinant plasmids and strains were also constructed to containpolynucleotide segments having the sequences as set forth in SEQ IDNO:9, SEQ ID NO:13, SEQ ID NO:21, and SEQ ID NO:23, and were confirmedto encode proteins having amino acid sequences as set forth in SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:22, and SEQ ID NO:24 (respectively,TIC1925, TIC1885, TIC2032, and TIC2120).

Example 3: Lepidopteran Activity of TIC1886

This example illustrates the Helicoverpa zea (Hz) activity exhibited bya sample from a recombinant strain expressing recombinant proteinTIC1886 having deduced amino acid sequence of SEQ ID NO:8.

A diet of 16.5% (w/v) of “Multiple Species” diet (Southland Products,201 Stuart Island Road, Lake Village, Ark. 71653) was prepared in a 14%(w/v) agar base (Serva #11393). The agar base was melted and blendedwith diet and purified water to volume (1.4% (w/v) agar). Dietsuspension was dispensed into individual bioassay compartments.

A test sample of recombinant protein TIC1886 was prepared as describedin example 2, and overlaid over 24 compartmentalized diet surfacesapproximating about 2890 ug/mL per compartment. A buffer control samplewas overlaid onto 96 compartmentalized diet surfaces. Together, 120compartmentalized diet surfaces comprise the test set of this exampleprepared for Helicoverpa zea bioassay.

A single neonate larva was transferred to the diet surface of eachindividual compartment of the test set of this example (120 totalneonates) and each compartment sealed with a ventilated cover. Thetest-set was placed in a controlled environment at 27° C. and 60% RHwith no light for 5-7 days and scored for mortality and stunting.Stunting was visually estimated in comparison to untreated insects andwas scored as significantly stunted (>67% stunting), moderately stunted(33-67% stunted), or stunted (<33%).

No stunting or mortality was observed with buffer control samples.Mortality was observed against 24 Hz larvae at 2890 ug/mL TIC1886. Itwas concluded that the protein TIC1886 demonstrated Helicoverpa zea(corn earworm) activity (see FIG. 2 and Table 2).

The lepidopteran bioassay procedure described in this example was alsoapplied to a combination of larvae from Ostrinia nubilalis, Diatraeasaccharalis, Diatraea grandiosella, and Anticarsia gemmatalis speciesusing the proteins of the present invention (from Example 2, TIC1498,TIC1415, TIC1497, TIC1414, TIC1422, and TIC1362), and the results aredescribed in examples 4-7.

Example 4: Lepidopteran Activity of TIC1498, TIC1497, and TIC1422

Using the methods and bioassay techniques described in Example 3,recombinant proteins TIC1498 (SEQ ID NO:2), TIC1497 (SEQ ID NOs:6), andTIC1422 (SEQ ID NO:18) were tested against neonates of Ostrinianubilalis (On), Diatraea saccharalis (Ds), Diatraea grandiosella (Dg),and Anticarsia gemmatalis (Ag) insect species.

TIC1498 exhibited mortality against Ostrinia nubilalis, and survivorswere significantly stunted, over 24 larvae at 2500 ug/mL TIC1497exhibited mortality against Ostrinia nubilalis, and survivors weresignificantly stunted, over 24 larvae at 3700 ug/mL. TIC1422 exhibitedmortality against Ostrinia nubilalis, and survivors were significantlystunted, over 24 larvae at 1300 ug/mL. It was concluded that TIC1498,TIC1497, and TIC1422 demonstrated activity (see FIG. 2 and Table 2)against Ostrinia nubilalis (European corn borer).

TIC1498 exhibited mortality against Diatraea saccharalis, and survivorswere significantly stunted, over 24 larvae at 3000 ug/mL. TIC1497exhibited mortality against Diatraea saccharalis, and survivors weresignificantly stunted, over 24 larvae at 2000 ug/mL. TIC1422 exhibited100% mortality to Diatraea saccharalis, over 24 larvae at 300 ug/mL. Itwas concluded that recombinant proteins TIC1498, TIC1497, and TIC1422demonstrated activity (see FIG. 2 and Table 2) against Diatraeasaccharalis (surgarcane borer).

TIC1498 exhibited mortality against Diatraea grandiosella, and survivorswere moderately stunted, over 24 larvae at 3000 ug/mL TIC1497 exhibitedmortality rate against Diatraea grandiosella, and survivors weresignificantly stunted, over 24 larvae at 2000 ug/mL. TIC1422 exhibitedmortality against Diatraea grandiosella, over 24 larvae at 300 ug/mL. Itwas concluded that TIC1498, TIC1497, and TIC1422 demonstrated activity(see FIG. 2 and Table 2) against Diatraea grandiosella (southwesterncorn borer).

TIC1498 exhibited mortality against Anticarsia gemmatalis, and survivorswere significantly stunted, over 48 larvae at 2500-3000 ug/mL. TIC1497exhibited mortality against Anticarsia gemmatalis, and survivors weremoderately to significantly stunted, over 48 larvae at 2000-3700 ug/mL.TIC1422 exhibited mortality against Anticarsia gemmatalis, and survivorswere significantly stunted, over 48 larvae at 300-1300 ug/mL. It wasconcluded that TIC1498, TIC1497, and TIC1422 demonstrated activity (seeFIG. 2 and Table 2) against Anticarsia gemmatalis (velvetbeancaterpillar).

Example 5: Lepidopteran Activity of TIC1415

TIC1415 (SEQ ID NO:4) was tested against Ostrinia nubilalis (On) andAnticarsia gemmatalis (Ag) insect species neonates. TIC1415 exhibitedmortality against Ostrinia nubilalis, and survivors were moderatelystunted, over 24 larvae at 1500 ug/mL. It was concluded that TIC1415demonstrated activity (see FIG. 2 and Table 2) against Ostrinianubilalis (European corn borer).

TIC1415 exhibited mortality against Anticarsia gemmatalis, and survivorswere moderately stunted, over 24 larvae at 1500 ug/mL. It was concludedthat TIC1415 demonstrated activity (see FIG. 2 and Table 2) againstAnticarsia gemmatalis (velvetbean caterpillar).

Example 6: Lepidopteran Activity of TIC1414

TIC1414 (SEQ ID NO:12) was tested against Anticarsia gemmatalis (Ag)insect species neonates. TIC1414 exhibited mortality against Anticarsiagemmatalis, and survivors were stunted, over 24 larvae at 870 ug/mL. Itwas concluded that TIC1414 demonstrated activity (see FIG. 2 and Table2) against Anticarsia gemmatalis (velvetbean caterpillar).

Example 7: Lepidopteran Activity of TIC1362

TIC1362 (SEQ ID NO:26) was tested against Diatraea saccharalis (Ds) andDiatraea grandiosella (Dg) insect species neonates. TIC1362 exhibitedmortality against Diatraea saccharalis, and survivors were significantlystunted, over 24 larvae at 400 ug/mL. TIC1362 demonstrated Diatraeasaccharalis (velvetbean caterpillar) activity (see FIG. 2 and Table 2).

TIC1362 exhibited 100% mortality against Diatraea grandiosella over 24larvae at 400 ug/mL. TIC1362 demonstrated Diatraea grandiosella(southwestern corn borer) activity (see FIG. 2 and Table 2).

Example 8: Hemipteran Activity of TIC1498

This example illustrates insect inhibitory activity of TIC1498 (SEQ IDNO:2) when provided in the diet of hemipteran insects, including but notlimited to members of the Heteroptera miridae, including the genusLygus, e.g., Lygus hesperus and Lygus lineolaris. This example morespecifically illustrates the Lygus hesperus (Lh) and Lygus lineolaris(Ll) activity exhibited by a sample from a recombinant strain expressingthe recombinant protein TIC1498.

A diet of 7.81% (w/v) of “Lygus Diet” diet (Bio-Sery #F9644B, One 8thStreet, Suite One, Frenchtown, N.J. 08825) and liquid contents of twowhole fresh eggs was prepared. The diet was cooled and stored undermoisture controlled conditions and at 4° C. until ready for use. Thisdiet preparation was used within 2 days of preparation.

Test samples containing TIC1498 protein were prepared encapsulated (˜40uL) between stretched Parafilm and Mylar sheets that were heat-sealed(sachets).

Lygus hesperus and Lygus lineolaris eggs were incubated at 24° C. untilthey reached between 0 to about 12 hours pre-hatch stage. Pre-hatch eggswere soaked and rinsed in sterile water, then placed in confinedproximity to the prepared sachets in a controlled environment at 24° C.and 60% RH with no light for 4-7 days and scored for percent mortalityand stunting of any survivors. Stunting was visually estimated incomparison to untreated insects and was scored as significantly stunted(>67% stunting), moderately stunted (33-67% stunted), or stunted (<33%).

At 10 ug/mL TIC1498, mortality was observed against Lygus lineolaris,and survivors stunted, over 24 neonate nymphs. At 50 ug/mL TIC1498,mortality was observed against Lygus lineolaris, and survivors stunted,over 24 neonate nymphs. TIC1498 exhibited mortality against Lyguslineolaris at 100 ug/mL, and survivors were moderately stunted, over 24neonate nymphs.

At 10 ug/mL TIC1498, mortality was observed against Lygus hesperus, andsurvivors stunted, over 24 neonate nymphs. At 50 ug/mL TIC1498,mortality was observed against Lygus hesperus, and survivors stunted,over 24 neonate nymphs. TIC1498 exhibited mortality against Lygushesperus at 100 ug/mL, and survivors were moderately stunted, over 24neonate nymphs. At 2300 ug/mL TIC1498, 100% mortality was observedagainst Lygus hesperus, over 24 neonate nymphs.

TIC1498 demonstrated both Lygus lineolaris (tarnished plant bug) andLygus hesperus (Western tarnish plant bug) activity (see FIG. 2 andTable 2). The hemipteran bioassay procedure described in this examplewas also performed using TIC1415, TIC1497, TIC1886, TIC1414, TIC1922,TIC1974, and TIC1362.

Example 9: Hemipteran Activity of TIC1922 and TIC1974

TIC1922 (SEQ ID NO:16) and TIC1974 (SEQ ID NO:20) were tested againstLygus lineolaris (Ll). TIC1922 was tested in 3 groups of 24 sachets, andexhibitedmortality against Lygus lineolaris, and survivors exhibitedstunting at 3000 ug/mL TIC1974 did not exhibit mortality against Lyguslineolaris, but survivors were stunted, in an evaluation of 24 neonatenymphs at 3000 ug/mL. TIC1922 and TIC1974 demonstrated Lygus lineolaris(tarnished plant bug) activity (see FIG. 2 and Table 2).

Example 10: Hemipteran Activity of TIC1497

TIC1497 (SEQ ID NO:6) was tested against Lygus hesperus (Lh). TIC1497exhibited mortality against Lygus hesperus, and survivors weremoderately stunted, in an experiment evaluating 48 neonate nymphs at2000 ug/mL.

Preparations of TIC1497 fragments were made by treating TIC1497 withthermolysin, chymotrypsin, trypsin, or Glu-C, resulting in TIC1497fragments exhibiting masses of 32411 Da (SEQ ID NO:64), 32557 Da (SEQ IDNO:62), 34225 Da (SEQ ID NO:61), and 34485 Da (SEQ ID NO:63). Theprotein eluate from the thermolysin treated preparation was isolated(TIC1497.32411) on an ion exchange column and used in bioassays againstHemipteran species.

TIC1497.32411 exhibited mortality against Lygus lineolaris, andsurvivors were moderately stunted, in an experiment using 24 neonatenymphs at 100 ug/mL. TIC1497.32411 exhibited 100% mortality at a dose of1000 ug/mL.

TIC1497.32411 exhibited mortality against Lygus lineolaris, andsurvivors were moderately stunted, in an experiment using 24 neonatenymphs at a dose of 2300 ug/mL.

TIC1497 demonstrated Lygus hesperus (Western tarnish plant bug) activity(see FIG. 2 and Table 2). The fragment TIC1497.32411 demonstrated bothLygus lineolaris (tarnished plant bug) and Lygus hesperus (Westerntarnish plant bug) activity (see FIG. 2 and Table 2).

Example 11: Hemipteran Activity of TIC1886, TIC1415, TIC1414, andTIC1362

TIC1886 (SEQ ID NO:8), TIC1415 (SEQ ID NO:4), TIC1414 (SEQ ID NO:12),and TIC1362 (SEQ ID NO:24) were tested against Lygus lineolaris andLygus hesperus. TIC1886 exhibited mortality against both Lyguslineolaris and Lygus hesperus, at a dose equivalent to 124 ug/mL.TIC1415 exhibited mortality against Lygus lineolaris and Lygus hesperusat a dose equivalent to 150 ug/mL and survivors were stunted. TIC1414exhibited mortality against Lygus lineolaris at a dose equivalent to 95ug/mL. TIC1362 exhibited mortality against Lygus lineolaris at a doseequivalent to 370 ug/mL.

TIC1886, TIC1415, and TIC1362 demonstrated both Lygus lineolaris(tarnished plant bug) and Lygus hesperus (Western tarnish plant bug)activity. TIC1414 demonstrated Lygus lineolaris (tarnished plant bug)activity (see FIG. 2 and Table 2)

Example 12: Insect Inhibitory Activities of Other Protein Members

Other protein members from the genus of the present invention, such asbut not limited to TIC1925 (SEQ ID NO:10), TIC1885 (SEQ ID NO:14),TIC2032 (SEQ ID NO:22), and TIC2120 (SEQ ID NO:24), are prepared forbioassay against pests of plants, including a pest from the phylumNematoda, a pest from Lepidoptera, and a pest from Hemiptera.

Example 13: Protein Expression in Plants

This example illustrates expression of proteins of the present inventionin plants. Polynucleotide segments for use in expression of the proteinsof the present invention in plants can be produced according to themethods set forth in U.S. Pat. No. 7,741,118. For example, toxinproteins having the amino acid sequence as set forth in SEQ ID NO:4, SEQID NO:12, SEQ ID NO:18, and SEQ ID NO:26 can be produced in plants frompolynucleotide segments having the sequence as set forth respectively inSEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.Polynucleotide segments designed for use in plants and encoding theproteins of the present invention, including the sequences as set forthin SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 areoperably linked to the requisite expression elements for expression inplants, and transformed into the genome of plant cells, preferablycotton, alfalfa, corn, and soybean cells.

It is intended that polynucleotide segments (or polynucleotidemolecules) encoding each of the following enumerated proteins, insectinhibitory fragments thereof, and proteins exhibiting the degree ofidentity specified herein above to one or more of these enumeratedproteins, be used alone or in combinations with each other, or incombinations with other toxin proteins or toxic agents such as dsRNAmediated gene suppression molecules designed to work in synergistic orsynonymous ways with the proteins of the present invention, to achieveplants and plant cells protected from pest infestation, particularlyinsect pest infestation. The specific enumerated proteins within thescope of the invention include TIC1498 (SEQ ID NO:2), TIC1415 (SEQ IDNO:4), TIC1497 (SEQ ID NO:6), TIC1886 (SEQ ID NO:8), TIC1925 (SEQ IDNO:10), TIC1414 (SEQ ID NO:12), TIC1885 (SEQ ID NO:14), TIC1922 (SEQ IDNO:16), TIC1422 (SEQ ID NO:18), TIC1974 (SEQ ID NO:20), TIC2032 (SEQ IDNO:22), TIC2120 (SEQ ID NO:24), TIC1362 (SEQ ID NO:26), TIC2335 (SEQ IDNO:136), TIC2334 (SEQ ID NO:138) and insect inhibitory fragmentsthereof, such as but not limited to, TIC1497.34225 (SEQ ID NO:61),TIC1497.32557 (SEQ ID NO:62), TIC1497.34485 (SEQ ID NO:63), andTIC1497.32411 (SEQ ID NO:64).

For instance, proteins of the TIC1415 genus of the present invention canbe combined with other pesticidal agents, including pesticidal agentstargeting pests which overlap with pests targeted by TIC1415 proteins.Additionally, other pesticidal agents may include agents that targetpests that do not overlap with pests targeted by TIC1415 proteins. Ineither case, it is intended that TIC1415 proteins be used alone orcombined with other pesticidal agents. In the examples described below,TIC1415 was co-expressed with a TIC807 toxin protein in cotton plantsand in planta bioassays were conducted. In addition to TIC807 toxinproteins, other pesticidal agents that can be used in combination withTIC1415 proteins include (1) hemipteran-centric agents, e.g. dsRNAdirected towards hemipteran orthologs of Nilaparvata lugens V-ATPase-E,21E01 (Li, Jie et al., 2011, Pest Manag Sci); dsRNA directed towardshemipteran orthologs of five different genes—actin ortholog, ADP/ATPtranslocase, α-tubulin, ribosomal protein L9 (RPL9) and V-ATPase Asubunit (Upadhyay, S. K., et al., 2011, J. Biosci. 36(1), p. 153-161);AXMI-171 (US20100298207A1); Bt endotoxins such as Cry3A, Cry4Aa,Cry11Aa, and Cyt1Aa, which were found to exhibit low to moderatetoxicity on the pea aphid, Acyrthosiphon pisum, in terms of bothmortality and growth rate (Porcar, M. et al., Applied and EnvironmentalMicrobiology, July 2009, p. 4897-4900, Vol. 75, No. 14); (2) otherColeopteran pesticidal agents, e.g. DIG11 and DIGS; Cry7; eCry3.1Ab;mCry3A; Cry8; Cry34/Cry35; and Cry3 toxins generally; and (3) otherLepidopteran pesticidal agents, e.g. DIG2; Cry1 toxins; Cry1A.105; Cry2toxins, particularly Cry2A toxins; Cry1F toxins; VIP3 toxins; and Cry9toxins. Transgenic crop events expressing other pesticidal agents canalso be used in combination with crop events expressing TIC1415proteins, examples of which include MON88017, MON89034, MON863,MON15985, MON531, MON757, COT102, TC1507, DAS59122-7, 3006-210-23,281-24-236, T304-40, GHB119, COT67B, MIR162; corn event 5307, and thelike. Such combinations with events expressing one or more proteins ofthe TIC1415 genus proteins provide more durable pest protection, providea resistance management strategy for target pest control, and reducefarmer inputs, saving considerable expense in time and monetary value.

Recombinant plants are generated from transformed plant cells of thisexample, and the recombinant plants or their progeny are evaluated forresistance to pest infestation, such as tolerance to Hemiptera and/orLepidoptera. Transgenic plants and seed are selected that provide pestresistance, such as to Hemiptera and Lepidoptera, and such plants andseed are advanced for further development.

Example 14: In-Planta Bioassay of TIC1415

In this study, toxin protein TIC1415 having the amino acid sequence asset forth in SEQ ID NO:4 was produced in plants from polynucleotidesegments having the sequence as set forth in SEQ ID NO:27. DNA havingthe sequence of SEQ ID NO:27 encoding TIC1415 was cloned into anAgrobacterium-mediated plant transformation vector along with requisitepromoter and regulatory elements for transformation and expression incotton cells. Transgenic cotton plants (recombinant cotton plants) wereproduced and tested for efficacy. Regenerated (R0) transgenic plantswere transferred to soil and tissue samples selected from transformationevents that were low in copy number and expressing TIC1415 protein.Lyophilized tissue samples of R0 plants from three events were weighedand combined 1:50 and 1:100 (weight:buffer) of 25 mM Sodium-carb/bicarbbuffered at pH 10.5 to extract soluble protein from the tissue. Sampleswere confirmed by Western blot for presence of TIC1415 protein. Sampleextracts were fed to Lygus lineolaris using the bioactivity assaydescribed in Example 8. Extract from DP393 cotton tissue absent ofTIC1415 protein was also prepared as negative control. Sample extractsfrom all three events exhibited mortality against Lygus lineolaris andsurvivors were stunted. Mortality and stunting scores were significantcompared to bioactivity scores of insects fed with sample extracts fromthe DP393 negative control.

R0 plants were grown and self-pollinated to obtain seed homozygous forthe introduced transgenic DNA. Homozygous plants from three differentsingle copy events were selected and five seed per event planted andevaluated in a whole plant caging assay. Plants were grown to floweringstage and each whole cotton plant was enclosed in a mesh cage made fromperforated plastic. Two pairs of male and female Lygus hesperus adultswere placed into each cage and allowed to reproduce. Resulting insectprogeny were allowed to infest the caged cotton plants for 3 weeks. Atthe end of the 21 day period, Lygus insects at various stages ofdevelopment were counted and average means calculated on a per plantbasis. Plants from all three events had significantly less insectscompared to the DP393 negative control. See Table 4.

TABLE 4 In-planta bioassay of TIC1415. Mean Mean Mean Mean Mean 3rd 4th5th Live 2nd Total 2nd Students t Instar Instar Instar Gen. Gen.Grouping Event or < Nymphs Nymphs Adults Lygus (p < 0.05) 84 0.20 0.000.60 0.00 0.80 B 52 0.00 0.40 1.60 1.20 3.20 B 39 0.40 0.40 2.00 1.604.40 B DP393 3.60 5.20 9.70 9.50 28.0  A (negative) Mean 2^(nd)Generation Lygus Recovered from Five Cotton Plants per Event in a CagedWhole Plant Assay. Events with the same letter do not have statisticallydifferent 2^(nd) generation Lygus numbers (p < 0.05, Students t).

Example 15: Protein Bioassay of TIC1415 and a TIC807 Hemipteran ToxicProtein

Protein samples were prepared containing various mixtures of TIC1415 anda TIC807 hemipteran toxic protein and tested in bioassay. TIC1415protein alone and the TIC807 protein alone were also prepared aspositive controls. Buffer was used as negative control. Sample mixtureswere fed to Lygus lineolaris using bioactivity assay. All threepreparations containing toxin protein exhibited mortality against Lyguslineolaris and survivors were stunted. Mortality and stunting scoreswere significant compared to bioactivity scores of insects fed withbuffer (see Table 5). The data suggests that there are no antagonisticeffects. Additional bioassay tests are performed on mixtures todemonstrate synergistic and/or additive effects.

TABLE 5 Bioassay data for protein mix: TIC1415 combined with a TIC807toxin protein Mean^(†) T Mean^(†) T TIC1415 TIC807 Population Groupingstunting^(‡) Grouping SAMPLE (ug/mL) (ug/mL) mortality on mort score onstunting TIC1415 + 4.35 1 21.79 AB* 0.60 AB* TIC807 TIC1415 + 2.175 120.36 B* 0.60 AB* TIC807 TIC1415 + 1.0875 1 12.50 BC 0.60 AB* TIC807TIC1415 + 4.35 0.5 32.50 A* 0.80 A* TIC807 TIC1415 + 1.75 0.265 7.86 CD0.40 ABC TIC807 TIC1415 + 0.875 0.265 0.00 D 0.00 C TIC807 TIC1415 +0.4375 0.265 5.36 CD 0.00 C TIC807 TIC1415 + 4.35 0.25 13.21 BC 0.40 ABCTIC807 TIC1415 + 1.75 0.1325 0.00 D 0.00 C TIC807 TIC1415 + 1.75 0.066250.00 D 0.00 C TIC807 TIC1415 4.35 0 12.50 BC 0.40 ABC TIC1415 1.75 07.86 CD 0.00 C TIC807 0 1 0.00 D 0.20 BC TIC807 0 0.265 2.50 CD 0.00 CBuffer (negative) 0 0 0.00 D 0.00 C control ^(†)Average (mean) of 5populations of 8 nymphs per population. ^(‡)Stunting scores correspondto visual mass ratings where 0 = no difference to negative control, 1 =about 25% less mass, 2 = about 50% less mass, and 3 = about 75% lessmass. The average of the stunting scores for each population of eightnymphs is reported. *At 95% confidence interval.

Example 16: In-Planta Bioassay of TIC1415 and TIC807

Transgenic cotton events were designed to co-express respective proteinsTIC1415 (SEQ ID NO:4) and a TIC807 protein. Such plants were evaluatedin a caged whole plant assay infested with Lygus lineolaris. Five plantseach from ten events were caged and infested with 2 pairs of male andfemale L. lineolaris per plant. The assay was incubated in a growthchamber under normal environmental conditions for cotton plantdevelopment for 21 days. DP393 negative control plants were grown insimilar manner. At the end of the 3 week period, Lygus of various stagesof development were counted. The mean number per plant of Lygus hesperusinsects at each stage in development were calculated and the results areshown in Table 6. Therein, different plant-expressible promoters wereused to drive expression of the transcript encoding TIC1415 in therespective constructs 12 and 13.

TABLE 6 In-planta data for protein mix: TIC1415 combined with a TIC807protein toxin Mean Mean Mean Mean Mean 3rd 4th 5th Live Total InstarInstar Instar 2nd Gen. 2nd Gen. Tukey Construct Event N or < NymphsNymphs Adults Lygus SEM Grouping 12 021 5 0.00 0.00 0.00 0.00 0.00 0.00B 625 5 0.20 0.20 0.20 0.00 0.60 0.24 B 830 5 2.20 0.20 0.00 0.00 2.401.12 AB 890 5 4.40 0.00 0.20 0.00 4.60 2.62 AB 521 5 4.60 0.60 0.00 0.005.20 4.27 AB 980 5 3.40 1.20 1.20 0.00 5.80 4.86 AB 13 426 5 0.00 0.000.00 0.00 0.00 0.00 B 611 5 0.60 0.00 0.00 0.00 0.60 0.60 B 999 5 0.400.00 0.40 0.00 0.80 0.37 B 356 5 6.20 0.00 0.40 0.00 6.60 4.73 AB DP3931 Inbred (Negative) 0 7.00 2.50 0.80 0.00 10.30  3.75 A Mean 2^(nd)Generation Lygus Recovered from Five Cotton Plants per Event in a CagedWhole Plant Assay. Events with the same letter do not have statisticallydifferent 2^(nd) generation Lygus numbers (p < 0.05, Students t).

What is claimed is:
 1. A recombinant nucleic acid molecule comprising aheterologous promoter operably linked to a polynucleotide segmentencoding an insect inhibitory polypeptide or an insect inhibitoryfragment thereof, wherein: (a) said insect inhibitory polypeptidecomprises an amino acid sequence having at least 80% amino acid sequenceidentity to SEQ ID NO: 26; or (b) said polynucleotide segment hybridizesunder stringent hybridization conditions to a polynucleotide having thenucleotide sequence of SEQ ID NO:
 25. 2. The recombinant nucleic acidmolecule of claim 1, wherein said insect inhibitory polypeptidecomprises an amino acid sequence having at least 90% identity to SEQ IDNO:
 26. 3. The recombinant nucleic acid molecule of claim 1, whereinsaid insect inhibitory polypeptide comprises an amino acid sequencehaving at least 95% identity to SEQ ID NO:
 26. 4. The recombinantnucleic acid molecule of claim 1, wherein said insect inhibitorypolypeptide comprises the amino acid sequence of SEQ ID NO:
 26. 5. Therecombinant nucleic acid molecule of claim 1 present within a host cell,wherein said host cell is selected from the group consisting of abacterial cell and a plant cell.
 6. The recombinant nucleic acidmolecule of claim 5, wherein said bacterial cell is a species from agenus selected from the group consisting of: Bacillus, Escherichia,Salmonella, Agrobacterium, Pseudomonas, and Rhizobium, and wherein saidBacillus species is a Bacillus thuringiensis, Bacillus subtilis,Bacillus megaterium, Bacillus cereus, or a Bacillus laterosperous. 7.The host cell of claim 5, wherein said plant cell is from a plantselected from the group consisting of alfalfa, banana, barley, bean,broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower,celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea,peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugar beet,sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass,tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. 8.The host cell of claim 5, wherein said plant cell is from a plant partselected from the group consisting of a seed, a boll, a leaf, a flower,a stem, and a root.
 9. The host cell of claim 5, wherein said host cellfurther comprises a herbicide tolerance marker.
 10. An insect inhibitorycomposition comprising a recombinant nucleic acid molecule comprising aheterologous promoter operably linked to a polynucleotide segmentencoding an insect inhibitory polypeptide or fragment thereof, and aninsect inhibitory polypeptide encoded by said polynucleotide segment, oran insect inhibitory fragment thereof, wherein: (a) said insectinhibitory polypeptide comprises an amino acid sequence having at least80% amino acid sequence identity to SEQ ID NO: 26; or (b) saidpolynucleotide segment hybridizes under stringent hybridizationconditions to a polynucleotide having the nucleotide sequence of SEQ IDNO:
 25. 11. The insect inhibitory composition of claim 10, wherein saidinsect inhibitory polypeptide exhibits insect inhibitory activityagainst a Hemipteran pest species.
 12. The insect inhibitory compositionof claim 11, wherein said Hemipteran pest species is selected from thegroup consisting of L. hesperus, L. lineolaris, A. hilare, E. servus, N.viridula, M. persicae, A. glycines, and A. gossypii.
 13. The insectinhibitory composition of claim 10, wherein said insect inhibitorypolypeptide exhibits insect inhibitory activity against a Lepidopteranpest species.
 14. The insect inhibitory composition of claim 13, whereinthe Lepidopteran pest species is selected from the group consisting ofH. zea, O. nubilalis, D. saccharalis, D. grandiosella, A. gemmatalis, S.frugiperda, S. exigua, A. ipsilon, T. ni, P. includens, H. virescens, P.xylostella, P. gossypiella, H. armigera, E. lignosellus, and P.citrella.
 15. The insect inhibitory composition of claim 10, furthercomprising at least one pesticidal agent, wherein said pesticidal agentis different from said insect inhibitory polypeptide, wherein saidpesticidal agent is selected from the group consisting of an insectinhibitory protein, an insect inhibitory dsRNA molecule, and anancillary protein.
 16. The insect inhibitory composition of claim 15,wherein said pesticidal agent exhibits insect inhibitory activityagainst the same Hemipteran species as said insect inhibitorypolypeptide, and wherein said pesticidal agent is selected from thegroup consisting of a TIC807 protein, a TIC853 protein, a Cry51 Aa1protein, and a AXMI-171 protein.
 17. A plant comprising the recombinantnucleic acid molecule of claim
 1. 18. A seed from the plant of claim 17,wherein said seed comprises said recombinant nucleic acid molecule. 19.A method of producing seeds comprising the recombinant nucleic acidmolecule of claim 1, said method comprising: (a) planting at least oneseed comprising the recombinant nucleic acid molecule of claim 1; (b)growing at least one plant from said at least one seed; (c) harvestingseeds from said at least one plant, wherein said harvested seedscomprise said recombinant nucleic acid molecule.
 20. A plant resistantto insect infestation, wherein cells of said plant comprise aninsecticidally effective amount of an insect inhibitory polypeptidecomprising an amino acid sequence having at least 80% amino acidsequence identity to SEQ ID NO:
 26. 21. A commodity product comprising adetectable amount of the insect inhibitory polypeptide of claim
 1. 22.The commodity product of claim 21, selected from the group consisting offlakes, cakes, flour, meal, syrup, oil, silage, starch, and cereal. 23.A method of controlling a Lepidopteran or Hemipteran pest species, saidmethod comprising contacting said Lepidopteran or Hemipteran pestspecies with an insect inhibitory amount of the insect inhibitorypolypeptide expressed by said recombination nucleic acid molecule ofclaim
 1. 24. The method of claim 23, wherein said contacting is viaexpressing said insect inhibitory recombinant polypeptide in a cropplant.